stahl experiment dna

The Meselson-Stahl Experiment (1957–1958), by Matthew Meselson and Franklin Stahl

In an experiment later named for them, Matthew Stanley Meselson and Franklin William Stahl in the US demonstrated during the 1950s the semi-conservative replication of DNA, such that each daughter DNA molecule contains one new daughter subunit and one subunit conserved from the parental DNA molecule. The researchers conducted the experiment at California Institute of Technology (Caltech) in Pasadena, California, from October 1957 to January 1958. The experiment verified James Watson and Francis Crick´s model for the structure of DNA, which represented DNA as two helical strands wound together in a double helix that replicated semi-conservatively. The Watson-Crick Model for DNA later became the universally accepted DNA model. The Meselson-Stahl experiment enabled researchers to explain how DNA replicates, thereby providing a physical basis for the genetic phenomena of heredity and diseases.

The Meselson-Stahl experiment stemmed from a debate in the 1950s among scientists about how DNA replicated, or copied, itself. The debate began when James Watson and Francis Crick at the University of Cambridge in Cambridge, England, published a paper on the genetic implications of their proposed structure of DNA in May 1953. The Watson-Crick model represented DNA as two helical strands, each its own molecule, wound tightly together in a double helix. The scientists claimed that the two strands were complementary, which meant certain components of one strand matched with certain components of the other strand in the double helix.

With that model of DNA, scientists aimed to explain how organisms preserved and transferred the genetic information of DNA to their offspring. Watson and Crick suggested a method of self-replication for the movement of genetic information, later termed semi-conservative replication, in which DNA strands unwound and separated, so that each strand could serve as a template for a newly replicated strand. According to Watson and Crick, after DNA replicated itself, each new double helix contained one parent strand and one new daughter strand of DNA, thereby conserving one strand of the original double helix. While Watson and Crick proposed the semi-conservative model in 1953, the Meselson-Stahl experiment confirmed the model in 1957.

In 1954, Max Delbrück at Caltech published a paper that challenged the Watson-Crick Model for DNA replication. In his paper, Delbrück argued that the replication process suggested by Watson and Crick was unlikely because of the difficulty associated with unwinding the tightly-wound DNA structure. As an alternative, Delbrück proposed that instead of the entire structure breaking apart or unwinding, small segments of DNA broke from the parent helix. New DNA, Delbrück claimed, formed using the small segments as templates, and the segments then rejoined to form a new hybrid double helix, with parent and daughter segments interspersed throughout the structure.

After the release of Delbrück´s paper, many scientists sought to determine experimentally the mechanism of DNA replication, which yielded a variety of theories on the subject by 1956. Delbrück and Gunther Stent, a professor at the University of California, Berkeley, in Berkeley, California, presented a paper in June 1956 at a symposium at Johns Hopkins University in Baltimore, Maryland, which named and summarized the three prevailing theories regarding DNA replication at the time: semi-conservative, dispersive, and conservative. Delbrück and Stent defined conservative replication as a replication mechanism in which a completely new double helix replicated from the parent helix, with no part of the parent double helix incorporated into the daughter double helix. They described the semi-conservative process as Watson and Crick suggested, with half of the parental DNA molecule conserved in the daughter molecule. Lastly, Delbrück and Stent summarized Delbrück´s dispersive model, in which parental DNA segments distribute throughout the daughter DNA molecule. Delbrück and Stent´s paper provided the background for the Meselson-Stahl experiment.

In 1954, prior to publication of Delbrück´s initial challenge of the Watson-Crick model, Matthew Meselson and Franklin Stahl had joined the DNA replication discussion. During the spring of 1954, Meselson, a graduate student studying chemistry at Caltech, visited Delbrück´s office to discuss DNA replication. According to historian of science Frederic Holmes, during that meeting Meselson began brainstorming ways to determine how DNA replicated. In the summer of 1954, Meselson met Stahl at the Marine Biological Laboratory in Woods Hole, Massachusetts. Stahl, a graduate student studying biology at the University of Rochester in Rochester, New York, agreed to study DNA replication with Meselson the following year at Caltech.

Meselson and Stahl began their collaboration in late 1956. By that time, Stahl had completed his PhD and Meselson had completed the experiments for his PhD, which he received in 1957. They worked on a variety of projects, including DNA replication. All of their projects, however, involved a method first devised by Meselson in 1954, called density-gradient centrifugation. Density-gradient centrifugation separates molecules based on their densities, which depend on the molecular weights of the molecules.

Meselson and Stahl used density-gradient centrifugation to separate different molecules in a solution, a method they later used to separate DNA molecules in a solution. In density gradient centrifugation, a solution is placed in an ultracentrifuge, a machine that spins the samples very fast on the order of 140,000 times the force of gravity or 44,770 revolutions per minute (rpm). As the samples spin, denser substances are pushed toward the bottom, while less dense substances distribute according to their weight in the centrifuge tube. By the end of centrifugation, the molecules reach a position called equilibrium, in which the molecules stop moving and remain in a gradient. The position of the molecules at equilibrium is dependent on the density of the molecule. Meselson and Stahl measured the areas in which DNA was at the highest concentration. Higher concentrations were represented by darker bands of DNA in the centrifuged sample. Stahl represented those bands on a graph, so that the peaks represented locations in the gradient where there was the highest concentration of molecules. Multiple peaks meant that molecules of different densities separated out of the solution.

To describe how DNA replicated, Meselson and Stahl needed to distinguish between parental and daughter DNA. They achieved that by modifying the molecules so each kind had a different density. Then Meselson and Stahl could separate the molecules using density-gradient centrifugation and analyze how much parental DNA was in the new daughter helices after every replication cycle. First they tried to alter the density of parental DNA by substituting a one nucleotide base, thymidine, with a heaver but similar DNA nucleotide base, 5-bromouriacil (5-BU). However, Meselson and Stahl struggled to substitute enough units of 5-BU into the DNA molecules to make the parental DNA significantly denser than normal DNA.

By July 1957, Meselson and Stahl successfully incorporated the heavy substitution in parental DNA, but the type of DNA they used still caused problems. Meselson and Stahl first used DNA from a specific type of virus that infects bacteria, called a bacteriophage. However, bacteriophage DNA not only broke apart in solution during centrifugation, but also replicated too quickly for the distribution of DNA to be adequately measured after each cycle. Consequently, Meselson and Stahl struggled to see clear locations within the density gradient with the highest concentration of bacterial DNA. Therefore, in September 1957, Meselson and Stahl switched to using the DNA from the bacteria Escherichia coli (E. coli) . E. coli DNA formed clearer concentration peaks during density gradient centrifugation.

At around the same time, in addition to changing the source DNA, Meselson and Stahl also changed the type of density label they used, from substitution labels to isotope labels. An isotope of an element is an atom with the same number of positive charged nuclear particles or protons, and a different number of uncharged particles, called neutrons. A difference in neutrons, for the most part, does not affect the chemical properties of the atom, but it alters the weight of the atom, thereby altering the density. Meselson and Stahl incorporated non-radioactive isotopes of nitrogen with different weights into the DNA of E. coli . As DNA contains a large amount of nitrogen, so long as the bacteria grew in a medium containing nitrogen of a specified isotope, the bacteria would use that nitrogen to build DNA. Therefore, depending on the medium in which E. coli grew, daughter strands of newly replicated DNA would vary by weight, and could be separated by density-gradient centrifugation.

Starting in October 1957, Meselson and Stahl conducted what later researches called the Meselson-Stahl experiment. They grew E. coli in a medium containing only the heavy isotope of nitrogen ( 15 N) to give the parental DNA a higher than normal density. As bacteria grow, they duplicate, thereby replicating their DNA in the process. The researchers then added an excess of light isotopes of nitrogen ( 14 N) to the heavy nitrogen environment.

Meselson and Stahl grew E. coli in the 14 N isotope environment for all subsequent bacterial generations, so that any new DNA strands produced were of a lower density than the original parent DNA. Before adding 14 N nitrogen, and for intervals of several bacterial generations after adding light nitrogen, Meselson and Stahl pulled samples of E. coli out of the growth medium for testing. They centrifuged each sample for initial separation, and then they added salt to the bacteria so that the bacteria released its DNA contents, allowing Meselson and Stahl to analyze the samples.

Next, Meselson and Stahl conducted density gradient centrifugation for each DNA sample to see how the parental and daughter DNA distributed according to their densities over multiple replications. They added a small amount of each sample of bacterial DNA to a cesium chloride solution, which when centrifuged had densities within the range of the bacterial DNA densities so that the DNA separated by density. The researchers centrifuged the DNA in an ultracentrifuge for twenth hours until the DNA reached equilibrium. Using ultraviolet light (UV), the researchers photographed the resulting DNA bands, which represented peaks of DNA concentrations at different densities. The density of the DNA depended on the amount of 15 N or 14 N nitrogen present. The more 15 N nitrogen atoms present, the denser the DNA.

For the bacterial DNA collected before Meselson and Stahl added 14 N nitrogen, the UV photographs showed only one band for DNA with 15 N nitrogen isotopes. That result occurred because the DNA from the first sample grew in an environment with only 15 N nitrogen isotopes. For samples pulled during the first replication cycle, the UV photographs showed fainter the 15 N DNA bands, and a new DNA band formed, which represented half 15 N DNA nitrogen isotopes and half 14 N DNA nitrogen isotopes. By the end of the first replication cycle, the heavy DNA band disappeared, and only a dark half 15 N and half 14 N DNA band remained. The half 15 N half 14 N DNA contained one subunit of 15 N nitrogen DNA and one subunit of 14 N nitrogen DNA. The data from the first replication cycle indicated some distribution of parental DNA, therefore ruled out conservative replication, because only parental DNA contained 15 N nitrogen isotopes and only parental DNA could represent the 15 N nitrogen isotopes in daughter DNA.

The same trends continued in future DNA replication cycles. As the bacteria continued to replicate and the bacterial DNA replicated, UV photographs showed that the band representing half 15 N half 14 N DNA depleted. A new band, representing DNA containing only 14 N nitrogen isotopes or light DNA, became the prevalent DNA band in the sample. The depletion of the half 15 N half 14 N band occurred because Meselson and Stahl never re-introduced 15 N nitrogen, so the relative amount of 15 N nitrogen DNA decreased. Meselson and Stahl then mixed the samples pulled from different replication cycles and centrifuged them together. The UV photograph from that run showed three bands of DNA with the half 15 N half 14 N DNA band at the midpoint between the 15 N DNA band and 14 N DNA band, making it an intermediate band. The result indicated that the half 15 N half 14 N DNA band had a density exactly between the 15 N and 14 N nitrogen DNA, showing that the DNA in the central band contained half of the 15 N nitrogen and half of the 14 N nitrogen isotopes, just as predicted by the Watson and Crick model. The exact split between heavy and light nitrogen characterized semi-conservative DNA replication.

Meselson and Stahl made three conclusions based on their results. First, they concluded that the nitrogen in each DNA molecule divided evenly between the two subunits of DNA, and that the subunits stayed intact throughout the observed replication cycles. Meselson and Stahl made that conclusion because the intermediate band had a density halfway between the heavy and light DNA bands. That conclusion made by Meselson and Stahl challenged the dispersive mechanism suggested by Delbrück, which involved breaking the DNA subunits into smaller pieces.

Meselson´s and Stahl´s second conclusion stated that each new DNA double helix contained one parental subunit, which supported semi-conservative replication. Assuming that DNA consists of two subunits, if a parent passes on one subunit of DNA to its offspring, then half of the parental DNA is conserved in the offspring DNA, and half of the parental DNA is not. The researchers made that conclusion because if parental DNA did not replicate in that way, then after the first replication, some DNA double helices would have contained only parental heavy nitrogen subunits or only daughter light nitrogen subunits. That type of replication would have indicated that that some parental DNA subunits did not separate in the semi-conservative fashion, and instead would have supported conservative replication. The presence of one parental subunit for each daughter DNA double helix supported semi-conservative replication.

The third conclusion made by Meselson and Stahl stated that for every parental DNA molecule, two new molecules were made. Therefore, the amount of DNA after each replication increased by a factor of two. Meselson and Stahl related their findings to the structure of DNA and replication mechanism proposed by Watson and Crick.

Before Meselson and Stahl published their findings, word of the Meselson-Stahl results spread throughout Caltech and the scientific community. According to Holmes, Delbrück, who had strongly opposed the semi-conservative method of DNA replication, immediately accepted DNA replication as semi-conservative after seeing the results from the Meselson-Stahl experiment. Some experiments earlier that year had pointed towards semi-conservative replication, and the Meselson-Stahl experiment served to further support semi-conservative replication.

Despite the positive reception of the Meselson-Stahl experiment, years passed before scientists fully accepted the Watson-Crick Model for DNA based on the findings from the Meselson-Stahl experiment. The Meselson-Stahl experiment did not clearly identify the exact subunits that replicated in DNA. In the Watson and Crick model, DNA consisted of two one-stranded DNA subunits, but the Meselson-Stahl experiment also supported models of DNA as having more than two strands. In 1959, Liebe Cavalieri, a scientist at the Sloan-Kettering Institute for Cancer research in New York City, New York, and his research team had produced evidence supporting the theory that DNA consisted of two two-stranded subunits, making DNA a quadruple helix. Cavalieri´s proposal did not contradict the Meselson-Stahl experiment, because the Meselson-Stahl experiment did not define DNA subunits. However, later experiments performed by Meselson on bacteriophage DNA from 1959 to 1961, and experiments performed by John Cairns on E. coli DNA in 1962, settled the debate and showed that each subunit of DNA was a single strand.

As described by Holmes, many scientists highly regarded the Meselson-Stahl experiment. Scientists including John Cairns, Gunther Stent, and James Watson all described the experiment as beautiful in both its performance and simplicity. Holmes also described the academic paper published by Meselson and Stahl on their experiment as beautiful because of its concise descriptions, diagrams, and conclusions. The Meselson-Stahl experiment appeared in textbooks decades after Meselson and Stahl performed the experiment. In 2001, Holmes published Meselson, Stahl, and the Replication of DNA: A History of "The Most Beautiful Experiment in Biology," which told the history of the experiment.

The Meselson-Stahl experiment gave a physical explanation for the genetic observations made before it. According to Holmes, for scientists who already believed that DNA replicated semi-conservatively, the Meselson-Stahl experiment provided concrete evidence for that theory. Holmes stated that, for scientists who contested semi-conservative replication as proposed by Watson and Crick, the Meselson-Stahl experiment eventually changed their opinions. Either way, the experiment helped scientists´ explain inheritance by showing how DNA conserves genetic information throughout successive DNA replication cycles as a cell grows, develops, and reproduces.

• Cairns, John. "A Minimum Estimate for the Length of the DNA of Escherichia coli Obtained by Autoradiography." Journal of Molecular Biology 4 (1962): 407–9.
• Cavalieri, Liebe F., Barbara Hatch Rosenberg, and Joan F. Deutsch. "The Subunit of Deoxyribonucleic Acid." Biochemical and Biophysical Research Communications 1 (1959): 124–8.
• Davis, Tinsley H. "Meselson and Stahl: The Art of DNA Replication." Proceedings of the National Academy of Sciences 101 (2004): 17895–6. http://www.pnas.org/content/101/52/17895.long (Accessed April 18, 2017).
• Delbrück, Max. "On the Replication of Deoxyribonucleic Acid (DNA)." Proceedings of the National Academy of Sciences 40 (1954): 783–8. http://www.pnas.org/content/40/9/783.short (Accessed April 18, 2017).
• Delbrück, Max and Gunther S. Stent. "On the Mechanism of DNA Replication." In McCollum-Pratt Symposium on the Chemical Basis of Heredity , eds. William D. McElroy and Bentley Glass, 699–736. Baltimore: Johns Hopkins University Press, 1956.
• Holmes, Frederic L. Meselson, Stahl, and the Replication of DNA: a History of "The Most Beautiful Experiment in Biology." New Haven: Yale University Press, 2001.
• "Interview with Matthew Meselson." Bioessays 25 (2003): 1236–46.
• Judson, Horace Freeland. The Eighth Day of Creation: Makers of the Revolution in Biology . Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1996.
• Levinthal, Cyrus. "The Mechanism of DNA Replication and Genetic Recombination in Phage." Proceedings of the National Academy of Sciences 42 (1956): 394–404. http://www.pnas.org/content/42/7/394.short (Accessed April 18, 2017).
• Litman, Rose M. and Arthur B. Pardee. "Production of Bacteriophage Mutants by a Disturbance of Deoxyribonucleic Acid Metabolism." Nature 178 (1956): 529–31.
• Meselson, Matthew. "The Semi-Conservative Replication of DNA." iBioMagazine 5 (2011). https://www.ibiology.org/ibiomagazine/issue-5/matthew-meselson-the-semi-conservative-replication-of-dna.html (Accessed April 18, 2017).
• Meselson, Matthew, and Franklin W. Stahl. "The Replication of DNA in Escherichia Coli." Proceedings of the National Academy of Sciences 44 (1958): 671–82. http://www.pnas.org/content/44/7/671.long (Accessed April 18, 2017).
• Meselson, Matthew, and Jean Weigle. "Chromosome Breakage Accompanying Genetic Recombination in Bacteriophage." Proceedings of the National Academy of Sciences 47 (1961): 857–68. http://www.pnas.org/content/47/6/857.short (Accessed April 18, 2017).
• Meselson, Matthew, Franklin W. Stahl, and Jerome Vinograd. "Equilibrium Sedimentation of Macromolecules in Density Gradients." Proceedings of the National Academy of Sciences 43 (1957): 581–8. http://www.pnas.org/content/43/7/581.short (Accessed April 18, 2017).
• Taylor, J. Herbert, Philip S. Woods, and Walter L. Hughes. "The Organization and Duplication of Chromosomes as Revealed by Autoradiographic Studies Using Tritium-Labeled Thymidine." Proceedings of the National Academy of Sciences 43 (1957): 122–8. http://www.pnas.org/content/43/1/122.short (Accessed April 18, 2017).
• Watson, James D., and Francis H C Crick. "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid." Nature 171 (1953): 737–8. https://profiles.nlm.nih.gov/ps/access/SCBBYW.pdf (Accessed April 18, 2017).
• Watson, James D., and Francis H C Crick. "Genetical Implications of the Structure of Deoxyribonucleic Acid." Nature 171 (1953): 964–7. https://profiles.nlm.nih.gov/ps/access/SCBBYX.pdf (Accessed April 18, 2017).
• Weigle, Jean, and Matthew Meselson. "Density Alterations Associated with Transducing Ability in the Bacteriophage Lambda." Journal of Molecular Biology 1 (1959): 379–86.

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How DNA Replicates

Matthew Meselson Franklin W. Stahl

Matthew Meselson had a passion for physics and chemistry throughout his early life, often conducting science experiments in his family's garage. At the age of 16, he enrolled at the University of Chicago, beginning an academic career that led to doctoral studies at the California Institute of Technology under Linus Pauling. In addition to his widely known work demonstrating semi-conservative replication of DNA with Frank Stahl, Meselson has made many key discoveries in the molecular biology. He is also known for his work in limiting the proliferation of chemical and biological weapons. Meselson is a member of the National Academy of Science and a recipient of the Lasker Award. He continues to serve as a member of the faculty at Harvard University, where he has taught and conducted research since 1960.

Following a sheltered life in a Boston suburb (Needham), Frank stumbled his way through college (Harvard, 1951) before fleeing to a graduate school in biology (U Rochester) to avoid the military draft. While in the graduate school, Frank took a course taught by A. H. (Gus) Doermann, and, for the first time in his life, he had a goal. With Gus, he studied genetic recombination in phage. To meet a departmental requirement, Frank took a summer course at Woods Hole, where he met Matt Meselson and began the work described in this Key Experiment. In 1959, Frank joined the faculty at the University of Oregon, Eugene, in their new Institute of Molecular Biology. He has been there ever since. Frank is now an emeritus faculty member who still enjoys teaching as well as family life and the natural wonders of Oregon.

What's the Big Deal?

Some experiments have proven so influential that they have been christened with the names of the scientists who performed them. The "Meselson–Stahl experiment" is one of those. It has also been called "the most beautiful experiment in biology," a title that has seemed to stick over the years. Why was the Meselson and Stahl experiment so important? Their experiment provided the first critical test of the Watson–Crick models for the structure of DNA and its replication, which were not universally accepted at the time. The convincing results of the Meselson–Stahl experiment, however, dispelled all doubts. DNA was no longer just an imaginary model; it was a real molecule, and its replication could be followed in the form of visually compelling bands in an ultracentrifuge. Meselson and Stahl found that these DNA bands behaved in the ultracentrifuge exactly as Watson and Crick postulated they should. Why was the Meselson–Stahl experiment "beautiful"? Because it was conceptually simple and yet sufficiently powerful to differentiate between several competing hypotheses for how DNA might replicate. Taken together, the Watson–Crick model and the Meselson–Stahl experiment marked the transition to the modern era of molecular biology, a turning point as impactful as the theory of evolution. The story of the Meselson–Stahl experiment, as told here by its protagonists, also reveals how friendship and overcoming obstacles are as crucial to the scientific process as ideas themselves.

Learning Overview —

Big concepts.

Faithful replication of the genetic material (DNA) is the foundation of all life on earth. The experiment by Meselson and Stahl established that DNA replicates through a semi-conservative mechanism, as predicted by Watson and Crick, in which each strand of the double helix acts as a template for a new strand with which it remains associated, until the next replication.

Bio-Dictionary Terms Used

Bacteriophage (phage) , base , base pairing , chromosome , DNA , Hershey–Chase experiment , eukaryote , mutation , nucleotides , Prokaryote (bacteria) , recombination , RNA , ultraviolet light

Terms and Concepts Explained

Equilibrium density-gradient centrifugation , DNA replication , isotope , semi-conservative DNA replication

Introduction

Matthew Meselson and Franklin Stahl (both 24 years old) met at the Marine Biological Laboratory in Woods Hole in Massachusetts and decided to test the Watson–Crick model for DNA replication, which was unproven at the time.

What Events Preceded the Experiment?

Watson and Crick proposed a "Semi-Conservative" model for DNA replication in 1953, which derived from their model of the DNA double helix. In this proposal, the strands of the duplex separate and each strand serves as a template for the synthesis of a new complementary strand. Watson's and Crick's idea for DNA replication was a model, and they did not have data to support it. Some prominent scientists had doubts.

Two other models, "Conservative" and "Dispersive", for DNA replication were proposed.

Setting Up the Experiment

A method was needed to detect a difference between the parental and daughter (newly replicated) DNA strands. Then, one could follow the parent DNA molecule in the progeny. Meselson thought to distinguish between parental and newly synthesized DNA using a density difference in the building blocks (nucleotides) used to construct the DNA. The three models for DNA replication would predict different outcomes for the density of the replicated DNA in the first- and second-generation daughter cells.

The general experimental idea was first to grow bacteria in a chemical medium to make high-density DNA and then abruptly shift the bacteria to a low-density medium so that the bacteria would now synthesize lower density DNA during upcoming rounds of replication. The old and newly synthesized DNA would be distinguished by their density.

To measure a density difference in the DNA, Meselson and Stahl invented a method called equilibrium density gradient centrifugation. In this method, the DNA is centrifuged in a tube with a solution of cesium chloride. When centrifuged, the cesium chloride, being denser than water, forms a density gradient, reaching a stable equilibrium after a few hours. The DNA migrates to a point in the gradient where its density matches the density of the CsCl solution. Heavy and light DNA would come to different resting points and thus physically separated.

Doing the Key Experiment

Meselson and Stahl first decided to study the replication of DNA from a bacteriophage, a virus that replicates inside of bacteria, and used a density difference between two forms of the nucleobase thymine (normal thymine and 5-bromouracil). These experiments did not work.

The investigators changed their plans. They studied replication of the bacterial genome and used two isotopes of nitrogen (15N (heavy) and 14N (light)) to mark the parental and newly synthesized DNA.

When the population of bacteria doubled, Meselson and Stahl noted that the DNA was of an intermediate density, half-way between the dense and light DNA in the gradient. After two doublings, half of the DNA was fully light and the other half was of intermediate density. These results were predicted by the Semi-Conservative Model and are inconsistent with the Conservative and Dispersive Models.

Meselson and Stahl did another experiment in which they used heat to separate the two strands of the daughter DNA after one round of replication. They found that one strand was all heavy DNA and the other all light. This result was consistent with the Semi-Conservative model and provided additional evidence against the Dispersive Model.

Overall, the results provided proof of Semi-Conservative replication, consistent with the model proposed by Watson and Crick.

What Happened Next?

Within a couple of weeks after their key experiment, Meselson wrote a letter to Jim Watson to share news of their result (letter included).

Max Delbruck, the Caltech physicist and biologist who had proposed the dispersive model, was elated by the results, even though Meselson and Stahl disproved his replication hypothesis, and urged the young scientists to write up their results for publication and announce the important result to the world (1958).

Scientists now know a great deal about the protein machinery responsible for DNA replication.

Closing Thoughts

The Meselson–Stahl experiment had a powerful psychological effect on the field of genetics and molecular biology. It was the first experimental test of the Watson and Crick model, and the results clearly showed that DNA was behaving in cells exactly as Watson and Crick predicted.

In addition to having a good idea, the behind-the-scenes tour of the Meselson–Stahl experiment reveals that friendship and persistence in overcoming initial failures play important roles in the scientific discovery process. Also important was an atmosphere of freedom that allowed Meselson and Stahl, then very junior, to pursue their own ideas.

Guided Paper

Meselson, M. and Stahl, F.W. (1958). The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences U.S.A., 44: 672–682.

This classic paper provides experimental evidence that the strands of the DNA double helix serve as templates to create a new copy of DNA. These results provide experimental evidence of The Watson and Crick model of DNA replication (‘semi-conservative replication’) demonstrating that genetic information is passed from one cell or organism to its progeny.

The first conversation between Matt Meselson and Frank Stahl, in the summer of 1954, began a collaboration that led to their Key Experiment on DNA replication and marked the beginning of a lifelong friendship. Matt and Frank describe the circumstances that brought them together below.

It was 1954, the year after Jim Watson and Francis Crick published their two great papers describing their double helical model of DNA and its implications for how it might replicate, mutate, and carry genetic information. Jim Watson (26 years old) and I (a 24-year-old first-year graduate student) were both at Caltech and living at the Athenaeum, the Caltech faculty club. We often talked while waiting for dinner. One day, Jim asked me to join him for the summer as his teaching assistant in the Physiology Course at the Marine Biological Laboratory (MBL) at Woods Hole, Massachusetts. He mainly wanted me to do experiments to see if RNA was a double helix. (As a side note, those experiments showed that RNA is not a double helix.) So in June 1954, I drove my 1941 Chevrolet coupe across the country from Cal Tech in Pasadena, California, to the MBL at Woods Hole, Massachusetts.

One day in Woods Hole, while planning student assignments for the Physiology Course, Jim went to the window of the course office upstairs in the MBL Lillie building and pointed towards a student sitting on the grass under a tree serving gin and tonics. That student was Frank Stahl. Jim said let's give him a really hard experiment to do all by himself – the Hershey–Chase Experiment; then, we'll see how good he really is. Two years earlier, Alfred Hershey and Martha Chase published an influential experiment that provided evidence implicating DNA, and not protein, as the substance conferring genetic inheritance in bacteriophage (see the whiteboard animation video on the Hershey–Chase experiment ).

I thought that this guy serving gin and tonics must be an interesting fellow, so I went downstairs to meet him and let him know what was being planned for him. Frank was then a biology graduate student at the University of Rochester. I sat down on the grass under the tree, and we hit it off right away. We found that we had much to discuss. I was very impressed with Frank's knowledge of phage genetics, a subject I knew nothing about, coming from the laboratory of Linus Pauling where I was doing X-ray crystallography. Frank can tell you more about our conversation under the "gin and tonic tree."

1954 was an exciting time for molecular biology. One year earlier, Watson and Crick published their model for the double helix structure of DNA (see the Narrative on DNA Structure by Vale ), which aroused much excitement as well as some serious disbelief. With Watson as an instructor and Crick and Sydney Brenner as visitors and several other founders of molecular biology, Woods Hole that summer became an epicenter for discussion of the great questions in molecular biology. Could the double helix model, as Watson and Crick proposed, explain the replication of the genetic information? What is the "code" for reading out the nucleotide sequence of DNA and turning that into the sequence of amino acids in protein? Would RNA have a similar structure to DNA? However, at the time of my arrival, I had no idea that Woods Hole was hosting anyone, but me, interested in the big problems of modern, i.e., molecular, biology.

I was a 24-year-old biology graduate student at the University of Rochester and had come to the MBL to take the Physiology Course. To beat the heat and, perchance, to meet someone interesting, I invested in a bottle of gin and a 6-pack of tonic, found some ice, a thermos jug, and a tree and sat myself down in the shade. Matt Meselson was one of my first catches.

Our conversation under the "gin and tonic tree" was life-changing. After Matt warned me of Watson's planned test of my experimental aptitude, we talked about the work we were doing. I explained to him the problem in phage genetics on which I was working. Eventually our conversation turned to DNA replication. Neither of us was working on the problem at the time, although we were both keenly interested in it. It was perhaps the most important contemporary question in molecular biology.

At Caltech, Matt had already come up with an idea for how the mechanism of DNA replication might be studied by density labeling. But, as a physical chemist, he was unfamiliar with the methods of phage and bacterial biology that would be needed to conduct the actual experiments. So we decided to collaborate. I was planning to be a postdoctoral fellow at Caltech starting that September. If we could develop a method for measuring the density of DNA molecules and successfully apply it to the problem of DNA replication, we could establish whether the Watson–Crick model for DNA replication, and even the model of the structure itself, was correct or not.

We did not begin our collaboration immediately because Matt needed to finish his X-ray crystallography and I had made plans to do my postdoctoral work on bacterial genetics with Joe Bertani.

You can also hear Matt Meselson describing the Meselson–Stahl experiment in Video 1 .

The genetic material of eukaryotic cells is organized in the form of chromosomes , each a single linear, double-stranded DNA molecule ( Figure 1 ). Most prokaryotes (bacteria) have a single, circular chromosome ( Figure 1 ). All forms of life must replicate their DNA and, except for recombination and infrequent mutations, pass identical copies of their genetic material to their progeny. (See also the Whiteboard Video on Keeping Track of Your DNA .)

Based upon their model for the structure of DNA, Watson and Crick proposed that DNA replicates in a "Semi-Conservative" manner ( Figure 2 ). In this model, the two strands of the DNA double helix unwind and separate, and each "parent" strand serves as a template for the synthesis of a new "daughter" strand. The Watson–Crick base pairing (see the Narrative on DNA Structure by Vale ) of adenine with thymine and guanine with cytosine ensures that, except for rare copying errors, mutations, the information of the original double-stranded DNA molecule is preserved during the synthesis of the daughter strands. In the Semi-Conservative model, each daughter cell in the first generation would inherit one of the original DNA strands from the parent and a recently synthesized DNA strand. In the second generation, two of the granddaughters would be composed of all newly synthesized DNA and two granddaughters would have hybrid DNA (one parental strand and one newly synthesized strand).

While (spoiler alert) the Semi-Conservative Model turned out to be correct, it was far from a foregone conclusion. Before our experiment, several leading scientists questioned the Semi-Conservative Model (see Dig Deeper 1 for more information on their reservations) and proposed alternate models discussed below.

Explorer's Question: What do you imagine are the pros and cons of this model?

Answer: The beauty of this model is that it provides a clear explanation of how a daughter strand is made from the template strand ( Figure 3 ). However, the model requires that the two long parental DNA strands unwind to become single-strand templates. This was seen as a weakness by many scientists at the time (see Dig Deeper 1 for more information).

The unwinding of the DNA helix and keeping the daughter and parental strands from becoming tangled posed problems for the Semi-Conservative model in the minds of many scientists. As a result, other models for DNA replication were imagined. One was a "Conservative Replication" model ( Figure 4 ). In this model, the parental double helix forms a template for a completely new double helix. The two original strands remain together, no unwinding occurs, and the daughter DNA is formed from newly synthesized DNA. In this model, in the first generation, one daughter DNA would inherit the original DNA double helix from the parent DNA and the other daughter DNA would inherit the newly synthesized DNA double helix. In the second generation, one of the four granddaughters would have the original parental DNA and the other three granddaughters would all be composed of newly synthesized DNA. While Conservative Replication was a logical possibility, it was not elaborated by any specific mechanism.

Answer: This model did not call for unwinding of the DNA strands, as in the Semi-Conservative Model, thus solving the concern about DNA unwinding. However, it was unclear how the copying machinery would read out the nucleotide sequence information buried in the core of the DNA double helix.

A third possibility was a model proposed by Max Delbrück (later called "Dispersive Replication") ( Figure 5 ). Delbrück doubted that the two strands of the double helix could be unwound or pulled apart to undergo Semi-Conservative replication and instead suggested that DNA strands broke at every half-turn of the helix during replication (discussed in more depth below and in Dig Deeper 1 ). According to Delbrück's Dispersive Replication Model, each helix of the replicated DNA consists of alternating parental and daughter DNA. Unlike the other two models, the progeny in subsequent generations would be indistinguishable with regard to their compositions of parental and newly synthesized DNA.

Answer: Fragmentation would create shorter templates for replication, which would minimize any unwinding or untangling problems faced by one very long DNA molecule. However, the reassembly of the fragments again into the intact chromosome could be problematic, especially if the breaks occur at every half-turn of the helix.

Explorer's Question: In the first generation, which model(s) would predict that the two daughter cells would receive approximately equal amounts of the original and newly synthesized DNA?

Answer: The Semi-Conservative Model and the Dispersive Model. However, differences in daughter composition arise in the second generation in the two models.

Matt and Frank learned about the models for DNA replication prior to their first meeting at the Physiology Course at the Marine Biological Laboratory through circumstances described below.

Sometime in 1953, while I was a graduate student of the great chemist Linus Pauling, I went to see Max Delbrück, a physicist and founder of the "phage group" who had become deeply interested in genetics and the basis of life (Max Delbrück, and his work with Salvador Luria, is featured in the Narrative on Mutations by Koshland ). I wanted to learn what problems in biology he thought were most important and what advice he might have for me about getting into biology. Almost as soon as I sat down in his office, he asked what I thought about the two papers by Watson and Crick that had been published in Nature earlier that year. I confessed that I had never heard of them.

Exasperated, Delbrück flung a little heap of reprints of the Watson–Crick papers at me and shouted "Get out and don't come back until you have read them." What I heard was "come back." So I did, but only after reading the papers.

When I came back, Delbrück said he did not believe in the Semi-Conservative mechanism of DNA replication proposed by Watson and Crick. Max had imagined that if replication is semi-conservative, the two daughter duplexes would become wound around each other turn-for-turn as the two chains of the mother molecule became unwound. To get around the supposed problem of untangling the daughter molecules, he proposed a model in which breaks are made to prevent interlocking when separating, and then joined back together (see Figure DD1 in Dig Deeper 1 ). This mechanism required rotation of only short lengths of duplex DNA, after which the chains would be rejoined. In the rejoining process, sections of the new chain would be fused to sections of the old chain, making all four of the chains mosaics of new and old DNA. Delbrück, in 1954, published a paper that questioned the Watson–Crick model and presented this new model (later referred to as "Dispersive Replication" as shown in Figure 5 and Dig Deeper 1 ). In some ways, the idea of Delbrück was ahead of its time. Transient breaks are now known to be made by an enzyme called topoisomerase, but in a manner that leaves the individual chains of the parent duplex intact (see Dig Deeper 1 ).

In addition to Max's reservations and model, several other scientists also posed their own concerns and solutions to the "unwinding problem" or alternatives to the Watson–Crick DNA structure itself (see Dig Deeper 1 ).

What I gathered from my conversations with Max and others was that not everyone believed the DNA replication model of Watson and Crick. It was only a hypothesis with no experimental evidence to support it. The key to solve this problem was to follow the parental DNA in the progeny. But how?

I was working on something very different for my PhD thesis with Linus Pauling, but earlier that year, I had an idea for labeling protein molecules with deuterium, a heavy isotope (2H) of hydrogen (1H) and separating them from unlabeled protein molecules in a centrifuge according to their density as a means to solve a quite different problem (see Dig Deeper 2 ). After that second meeting with Max, it occurred to me that density labeling and centrifugal separation might be used to solve the DNA replication problem. When I told this to Pauling, he urged me to get my X-ray crystallography done first. And when I proposed the density approach to Watson, one of those evenings waiting for dinner at the Athenaeum, he said I should go to Sweden to do it – where the ultracentrifuge had been invented (which I never did).

My entry point to thinking about DNA replication came when I was trying to understand how bacteriophage (viruses that infect bacteria) exchange pieces of DNA with one another. This process of DNA exchange between chromosomes is called recombination (see the whiteboard video on the experiments by Morgan and Sturtevant ). When did this recombination process occur? Did it occur when DNA replicates? Or perhaps recombination was an event that stimulated DNA replication? My intuition was that the processes of recombination and replication were somehow related. However, little was known about the mechanisms of either DNA replication or recombination at the time. Furthermore, I did not know how to pursue these questions in 1954. My ideas for experiments were lame and would have led to un-interpretable data.

Like many interesting questions in biology, often one has to be patient until either the right idea or technology emerges that allows one to answer them properly. In 1954, my awareness of a possible connection between replication and recombination primed my interest for the first gin and tonic conversation with Matt. However, it was several decades before I was sure that, in bacteriophage, DNA replication and recombination, in a large degree, depended upon each other. The convincing experiments were based on variations of a technique pioneered by Matt and Jean Weigle at Caltech. In this method, density-labeled, genetically marked parental phage infect the same bacteria. The densities and genetic makeup of progeny phage are determined by bioassay of the individual drops collected from a density gradient.

Matt and Frank

To distinguish between the Semi-Conservative, Conservative, and Dispersive Models of DNA replication described above, we needed a method that could tell the difference between the parental and daughter DNA strands. Figures 2 , 4 , and 5 illustrate the parent and newly synthesized strands with different colors. However, we needed to find a real physical difference that would serve the same function of distinguishing between the old and newly synthesized DNA. Matt had the idea of distinguishing old and new DNA by having the bacteria synthesize them with different isotopes and separating them in a centrifuge according to their density. If the original and the newly synthesized DNAs could be made of different density materials, then we could perhaps measure this physical difference. We will discuss the chemicals that were used to make the DNA heavier or lighter in the next section.

Our experimental idea was to grow an organism in a chemical medium that would make its DNA heavy. Then, while it was growing, we would switch to a new medium in which the newly synthesized DNA would be made of lighter material ( Figure 6 ). The density difference between the original and the newly replicated DNA could allow us to distinguish between models for DNA replication.

To separate DNA of different density, we invented a method, called "equilibrium density gradient centrifugation," and published it, together with Jerome Vinograd, a Caltech Senior Research Fellow who had taught us how to use the ultracentrifuge in his lab and provided advice. In this method, as applied to DNA, a special tube that has quartz windows so that ultraviolet light photos can be taken while the centrifuge is running is filled with a solution of cesium chloride and the DNA to be examined. Upon centrifugation at high speed (~45,000 revolutions per minute or 140,000 times gravity), the CsCl gradually forms a density gradient, becoming most concentrated at the bottom of the tube ( Figure 7 ). The CsCl solution toward the top of the tube is less dense than the DNA, while the CsCl solution at the bottom is denser than DNA. Thus, when a mixture of DNA in a CsCl solution is centrifuged, the DNA will eventually come to a resting point where its density matches that of the CsCl solution ( Figure 7 ). The DNA absorbs UV light, and its position along the tube was recorded by using a special camera while the centrifuge is running.

The method now seems straightforward, but in reality, it took a couple of years to develop. For example, we did not come to cesium chloride immediately. We looked at a periodic table for a dense monovalent atom that would not react with DNA; rubidium chloride (molecular weight of 121) was available in the Chemistry Department stockroom and we initially tried to use that but found that even concentrated solutions were not dense enough to float DNA to a banding point. We then moved one level down in the periodic table to cesium (the molecular weight of cesium chloride is 168) and that worked (for more details, see Dig Deeper 3 ).

Frank and Matt

In the fall of 1954, we were reunited in Cal Tech and lived for about eight months in the same house across the street from the lab. We finally could begin doing experiments to test models of DNA replication. It should be noted that DNA replication was our "side" project; we also had our "main" projects under the supervision of our respective professors. However, faculty at Cal Tech was kind in allowing two young scientists to venture forward with their own ideas.

While the general experimental approach that took form under the "gin and tonic tree" was straightforward, choices had to be made in how exactly to do the experiment. What organism should we use? Would a chemical trick of making DNA heavier or lighter work and could we measure a small density difference between the two? It took us a while to get the conditions right, about two years.

We first decided to examine the replication of the bacteriophage T4 inside of the bacterium Escherichia coli. Bacteriophages are viruses that invade and replicate inside of a bacterium; when new viruses are made, they will burst the bacterium and then spread to new hosts. Bacteriophage have small genomes and are therefore the smallest replicating systems. Frank's PhD thesis was on T4, so he knew how to work with this phage. Max Delbrück and others at Cal Tech were also actively studying phage (see the Narrative on Mutations by Koshland ). Thus, T4 seemed the obvious choice. To create DNA of heavier density than normal DNA, we decided to use the analogue, 5-bromouracil, of the base thymine, in which a heavier bromine atom replaces a lighter hydrogen atom. During replication, 5-bromouracil could be incorporated into DNA, instead of thymine.

However, while this approach seemed reasonable, it did not work in practice. Although we did not appreciate it at the time, during phage growth, the DNA molecules undergo recombination, joining parental DNA to newly synthesized DNA in a manner that after several generations would give no clear-cut distribution of old DNA among progeny molecules. Also, we learned from a recent paper that 5-bromouracil was mutagenic and made a detour into studying mutagenesis before coming back to our main project.

We clearly needed a new strategy.

Instead of phage, we decided to study the replication of the bacterial genome. This was a good choice – the bacterial DNA gave a very sharp and clear band when centrifuged in a solution of cesium chloride (to learn more about why we used cesium chloride to create a density gradient and the general use of this technique; see Dig Deeper 3 ).

We also switched our density label. DNA is made up of several elements – carbon, nitrogen, oxygen, phosphorus, and hydrogen. Some of these elements come in different stable isotopes, with atomic variations of molecular weight based upon different numbers of neutrons. Nitrogen-15 (15N) is a heavier isotope of nitrogen (the most common isotope, 14N, has a molecular weight of 14 Daltons). We could easily buy 15N in the form of ammonium chloride (15NH4Cl), which was the only source of nitrogen in our growth medium. The 15N in the medium then found its way into the bacterial DNA (as well as other molecules) in a harmless manner and did not impair bacterial growth.

We also had good luck in that Caltech bought a brand new type of ultracentrifuge called an analytical ultracentrifuge (Model E) developed by the Beckman Instrument Company. The Model E was a massive machine about the size of a small delivery truck (the current model is just a bit bigger than a dishwasher). Importantly, the Model E could shine a UV light beam on the tube while the centrifuge was spinning and detect and photograph the position of the DNA. The good news was that 15N-containing DNA and 14N-containing DNA could be clearly distinguished by their different density positions ( Figure 8 ).

Finally, we had everything in place to try our experiment properly. I decided to set up our first experiment in the following two ways:

1) Grow the bacteria in "light" nitrogen medium and then switch to "heavy."

2) Grow another culture of bacteria in "heavy" nitrogen for many generations and then switch to "light."

Frank was called to a job interview and could not perform this first experiment with me. But before leaving, he warned me – "Don't do the experiment in such a complicated way on your first try. You might mix up the tubes."

I ignored Frank's advice and did the experiment both ways.

In the first experiment after transferring bacteria grown in heavy nitrogen (15N) growth medium and then switched to "light" (14N) nitrogen medium, I saw three discrete bands corresponding to old, hybrid, and new DNA, as predicted by Semi-Conservative replication. Excited developing the photograph in the darkroom, I remember letting out a yelp that caused a young woman working nearby to leave in a hurry. But later I realized my mistake. Frank had been prophetic. I indeed had mixed tubes, combining two different samples, one taken before and the other taken after the first generation of bacterial growth in the light medium. As described for the correct experiment below, there is no time when old, hybrid, and fully new DNA are present at the same time.

When I came back from my trip, Matt and I performed what proved to be the decisive experiment. We grew bacteria in "heavy" nitrogen (15N; from 15NH4Cl) and then switched to "light" nitrogen (14N; 14NH4Cl) and, at different time points, collected the bacteria by centrifugation, added detergent to release the DNA, and combined this with concentrated CsCl solution to reach the desired density. After 20 hours of centrifugation and the final density positions of the DNAs had come into view, we knew that we had a clean answer ( Figure 9 ). The DNA from bacteria grown in heavy nitrogen formed a single band in the gradient. However, when the bacteria were shifted to a light nitrogen medium and then allowed to replicate their DNA and divide once (first generation), essentially all DNA had shifted to a new, "intermediate" density position in the gradient ( Figure 9 ). This intermediate position was half-way between the all heavy and all light DNA. At longer times of incubation in light nitrogen, after the cells had divided a second time (second generation), a DNA band at lighter density was seen and there were equal amounts of the intermediate and light DNA.

Explorer's Question: Which of the three models (Conservative, Semi-Conservative, or Dispersive) is most consistent with the results of this experiment?

Answer: The Semi-Conservative model. The Conservative model predicts a heavy and light band at the first generation, not an intermediate band. The Dispersive model predicts a single intermediate band at both the first and second generations (the band shifting toward lighter densities with more generation times).

Explorer's Question: Why are the two DNA bands at the 1.9 generation time point of approximately equal intensity?

Answer: After the first generation, each of the two heavy strands is partnered with a light strand. The bacterial DNA consists of one heavy strand and one light strand. When that heavy–light DNA replicates again in the light medium, the heavy strands are partnered with new light strands (intermediate density DNA) and the light strands are also partners with new light strands (creating all light density DNA).

The experiment that Frank described above took hardly any time at all (2 days) and yielded a clean result. We then repeated it without any problem. Once we knew how to set up the experiment, it was relatively easy. But it took us two years of trials before we got the experimental design and conditions right for the final ideal experiment.

The experiment clearly supported the Semi-Conservative Replication model for replication and, in doing so, also supported the double helical model of DNA itself. However, we wanted to do one more experiment that would examine whether the "intermediate" density band of DNA in the first generation was truly made of two and just two distinct subunits, as predicted by the Watson–Crick model. The model predicts that one complete strand of DNA is from the parent and should be heavy and the other complete DNA strand should be all newly replicated and therefore light ( Figure 10 ). We could test this hypothesis by separating the subunits with heat and then analyzing the density and molecular weight of the separated subunits by equilibrium density-gradient ultracentrifugation.

On the other hand, the Dispersive Model predicted that each DNA strand of first generation is an equal mixture of original and newly replicated DNAs ( Figure 11 ).

The results from the experiment were again clear ( Figure 12 ). The "intermediate density" DNA in the first generation split apart into a light and heavy component. From the width of the DNA band in the gradient (see Dig Deeper 3 ), we could also tell that the light and heavy DNA obtained after heating had each half of the molecular weight of the intermediate density DNA before heating. These results indicated that each parental strand remained intact during replication and produced a complete replica copy. This was decisive evidence against the Delbrück model for it predicted that both strands would be mosaics of heavy and light, not purely heavy and purely light. And the finding that the separated heavy and light subunits each had half the molecular weight of the intact molecule indicated that DNA was made up of two chains, as predicted by the Watson Crick model, and was not some multichain entity.

Based upon the results in Figure 9 and Figure 12 , we concluded that:

1) The nitrogen of a DNA molecule is divided equally between two subunits. The subunits remain intact through many generations.

2) Following replication, each daughter molecule receives one parental subunit and one newly synthesized subunit.

3) The replicative act results in a molecular doubling.

These conclusions precisely aligned with the Watson–Crick Semi-Conservative model for DNA replication. DNA, as a double-stranded helix, unwinds, and each strand serves as a template for the synthesis of a new strand.

When we had our result, Matt quickly shared the news with Jim Watson in a letter dated November 8, 1957 (available for the first time here ). It was common in those days to share results with colleagues through letters prior to a publication.

We also shared our results with Max Delbrück who took the news well that his Dispersive Replication model was incorrect. In fact, he wrote to a colleague that Meselson and Stahl had obtained a "world shaking result." But we were slow to get our work written up for publication. Once we knew the answer, we were keen to move onto new experiments rather than writing up our results.

Finally, Max had enough of our dallying and brought us down to the Caltech marine station at Corona del Mar. There, he quarantined us to a room in a tower, saying that we could not come out until we had written a draft of our paper. He was not being unkind, and we thought it great fun. Max's wife Manny Delbrück kindly came in occasionally to bring us delicious sandwiches, and Max also kept us company. We worked for 2 days straight and got him a draft.

Shortly thereafter, we completed our paper and Max communicated it in May 1958 to the Proceedings of the National Academies of Science, 4 years after our meeting at the Marine Biological Laboratory but less than a year after finally getting our experiments to work.

After our paper was published, we went separate ways in our lives. Frank got a job at the University of Missouri but soon thereafter moved to the University of Oregon in Eugene. Matt got promoted from a postdoctoral fellow to an assistant professor at Caltech and was teaching physical chemistry. However, the constant teaching limited time in the laboratory. Matt asked to be demoted from assistant professor back to senior postdoc, so he could get more work done in the lab. This is perhaps the only case in the history of Caltech in which a professor asked to move down the academic ladder. After a year as a senior postdoc, Matt then moved to Harvard to become an associate professor.

Decades have passed, and we now know much about the machinery that orchestrates DNA replication, including the unwinding of the strands and the synthesis of a new strand from the parental template. The details are beyond what can be discussed here, but you can view an animation of this process in Video 2 .

When we first discussed the use of density labeling to test models for DNA replication under the gin and tonic tree, we could not imagine the psychological effect our experiment would have on the field. Many scientists were not initially convinced by the Watson–Crick model for the structure of DNA or their proposal for its mode of replication. It was not clear if their model could explain heredity and the properties of genes. Some people seemed to think the model was too simple to be the gene. Others thought it too simple (meaning too beautiful) to be wrong!

However, after our experiment, the DNA model seemed very real. We could watch DNA with a camera; the visualization of DNA bands was simple and clear. Our results showed that the gene is made of two complementary halves, each a template for the other. Even the disbelievers, such as the deeply thoughtful Max Delbrück, acquiesced. DNA was no longer an imaginary molecule in the heads of Watson and Crick. It was a dynamic molecule; one could perform experiments on it, and it behaved in living cells as one might predict. Mendel's concept of a discrete "factor" that could determine a plant character and remain intact generation after generation and the physical reality of a gene as double-stranded DNA became intertwined from that moment on.

It is gratifying to think that our experiment, so simple by modern standards, is still valued and taught. But beyond the logic of how the experiment was performed, we hope that our story also conveys other important lessons about science.

• Every hypothesis needs to be rigorously tested with a clear experiment.

• An atmosphere of freedom is important. We were both very junior at the time of this experiment, but we were supported by senior scientists who encouraged us to pursue our own ideas.

• Success does not come immediately. Reading most scientific papers (including ours), everything seems straightforward and works right away. Our narrative shows that the so-called "most beautiful experiment in biology" had some unsuccessful excursions and two years of work to come to successful finish.

• Because success does not come immediately, it is valuable to be able to share difficult times with a friend. We kept each from getting discouraged. There was a certain gaiety in our work. We even had fun when things went wrong.

• Much of science is built upon collaboration and friendship. This Key Experiment could never have been the "Meselson Experiment" or the "Stahl Experiment." The "Meselson and Stahl Experiment" required both parties. We complemented each other scientifically and encouraged each other personally. Well more than a half-century has passed since this experiment was performed, and we remain good friends today.

Dig Deeper 1: Alternatives to Semi-Conservative replication

Max Delbrück, in his 1954 paper (PNAS 40: 783-788), said the following of the Watson and Crick Semi-Conservative replication mechanism:

"The principal difficulty of this mechanism lies in the fact that the two chains are wound around each other in a large number of turns and that, therefore, the daughter duplexes generated by the process just outlined are wound around each other with an equally large number of turns. There are three ways of separating the daughter duplexes: (a) by slipping them past each other longitudinally; (b) by unwinding the two duplexes from each other; (c) by breaks and reunions. We reject the first two possibilities as too inelegant to be efficient and propose to analyze the third possibility."

Max's solution was to break the single chains at regular intervals, allowing rotation about single bonds of the unbroken chain followed by joining in a way that dispersed short segments of parental DNA among the single chains of the daughter duplexes. This is the Dispersive Model presented in Figure 5 and presented in more detail in Figure DD1 . There was a germ of truth in Delbrück's idea of breakage. We now know of topoisomerases, enzymes that facilitate DNA replication by temporarily breaking, allowing unwinding, and then rejoining DNA. There are also enzymes that unwind DNA helixes called DNA helicases. Both enzymes use chemical energy derived from hydrolyzing adenosine triphosphate to perform work on the stable DNA double helix.

Another type of solution to the "unwinding problem," one that required no breaking and no entangling of the daughter molecules, was to imagine that the synthesis process would cause the entire parental duplex to rotate one turn for each turn of DNA synthesized. But this posed problems of its own – giving rise to a variety of long-forgotten proposed models, including evoking a motor at the growing point that would drive the rotation of the parent molecule, as proposed by John Cairns and Cedric Davern [J. Cellular Physiology, 70: S65–76 (1967)].

Alternative solutions questioned the DNA double helix model, but not semi-conservative replication. For example, one idea was to assume that the two chains are not wound around a common axis, but instead are simply pushed together (plectonemic coiling), which would require no unwinding and no rotation. This possibility, although it appeared remote, was not rigorously ruled out until much later in a paper by Crick, Wang, and Bauer in 1979 (J. Mol. Biol, 129: 449–461).

Dig Deeper 2: The idea for using density for separation

The idea for using density as a separation method came to me early in 1954 while I was a first-year graduate student at Caltech listening to a lecture by the great French scientist Jacques Monod. Monod was describing the problem of regulation of an enzyme called beta-galactosidase. If the bacteria were growing in a medium without lactose (a sugar), the enzyme activity was very low. When lactose or a chemical analogue of lactose was added, the enzyme activity was induced. The question was how? One model was that the enzyme was always there, but is inactive unless lactose is around. Another model (the correct one) was that the enzyme is synthesized de novo after the inducer is added. I thought that it might be possible to measure new enzyme and distinguish it from old enzyme if the enzyme was synthesized from heavier building blocks (amino acids). How could one make heavier building blocks? I thought that deuterium (a heavy isotope of hydrogen; 2 H) might be the answer. If one grew bacteria in heavy water ( 2 H 2 O) and switched to normal water ( 1 H 2 O) when one added inducer, then any newly synthesized beta-galactosidase would have had a greater density than the pre-existing beta-galactosidase. I never did the experiment, but the idea primed me for the DNA replication problem.

Dig Deeper 3: The role of the centrifuge in the Meselson–Stahl experiment

Matt describes briefly how this technique evolved

The first paper (see the reference list) that Frank and I wrote together (along with Jerome Vinograd) was on the method and theory of using centrifugation in an equilibrium density gradient, which showed that this method not only could separate molecules but could also be used as a tool to determine their molecular weights. This work was also part of my PhD thesis at Caltech. When I presented this work at my thesis defense, the great physicist Richard Feynman was on the examination committee, along with Pauling, Vinograd, and one of Pauling's post-docs who taught me X-ray crystallography. Feynman had not read the thesis but did so during the defense. I presented my rather long mathematical derivation showing that macromolecules in a density gradient in a centrifugal field would be distributed in a Gaussian manner about the position of neutral buoyancy with the width dependent upon the square root of the molecular weight. Feynman then went to the blackboard and, on the spot, produced a much shorter derivation of the same thing, modeled on the wave function for the quantum mechanical harmonic oscillator. Feynman writes about our experiment in his jolly book, Surely You're Joking, Mr. Feynman .

The way in which we found that CsCl forms a density gradient on its own was somewhat fortuitous. We initially thought that we needed to pour a CsCl gradient in the tube in advance. However, we found that just by centrifuging an initially homogeneous solution of cesium chloride produced a continuous gradient density on its own after several hours. From the width of the DNA band in the density gradient, we could also calculate the molecular weight of the DNA molecules in the gradient as 7 million Daltons. The chromosome of E. coli is much, much larger, but long molecules of DNA are fragile and had been broken up by shear forces while passing through the hypodermic needle with which we loaded the centrifuge cell. Subsequent to our result, CsCl equilibrium density-gradient centrifugation became a standard tool for isolating DNA from cells for decades and was used in important experiments such as the demonstration of messenger RNA by Brenner, Meselson, and Jacob and showing the mechanism of general recombination in phage lambda by Frank Stahl.

References and Resources

• Matthew Meselson’s letter to James Watson from November 8, 1957, describing the results of their experiments on DNA replication. Download .

This paper describes the use of the centrifuge and density gradient to analyze biological molecules, a technique that was used in their 1958 paper but also very broadly used for many applications in biology. See also Dig Deeper 3 .

An outstanding resource for those wanting a detailed, accurate description of the Meselson–Stahl experiment.

A nice 7:30 min video describing the Meselson–Stahl experiment and its conclusions.

This film documents the discovery of the structure and replication of DNA including interviews with James Watson who, along with Crick, proposed the double helix model of DNA.

This activity is often used in conjunction with the short film The Double Helix. It introduces students to Meselson and Stahl experiment and helps them understand the concepts generated via those experimental results.

This collection of resources from HHMI Biointeractive addresses many of the major concepts surrounding DNA and its production, reading, and replication.

• Illustrations
• Problem Sets
• Calculators

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Meselson–stahl experiment.

In their second paper on the structure of DNA * , Watson and Crick (pdf) described how DNA's structure suggests a pattern for replication:

"…prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before." - Watson and Crick, 1953

This is called semiconservative replication .

Today we know that this is the pattern used by living cells, but the experimental evidence in support of semiconservative replication was not published until 1958 . In the 5 years between Watson and Crick's suggestion and the definitive experiment, semiconservative replication was controversial and other patterns were considered.

Three hypothesized patterns were proposed:

• Semiconservative - The original double strand of DNA separates and each strand acts as a template for the synthesis of a complimentary strand.
• Conservative replication - the original double strand of DNA remains intact and is used as a template to create a new double stranded molecule.
• Dispersive replication - similar to conservative replication in that the original double strand is used as a template without being separated, but prior to cell division, the strands recombine such that each daughter cell gets a mix of new and old DNA. With each round of replication, the original DNA gets cut up and dispersed evenly between each copy.

The methods Meselson and Stahl developed allowed them to distinguish existing DNA from newly synthesized DNA and to track new and old DNA over several rounds of replication.

They accomplished this by labeling cells with different stable isotopes of nitrogen. First, a culture of bacterial cell were grown for several generations in a media containing only 15 N ( a stable, heavy isotope of Nitrogen). After this period * of growth, all of the DNA in the cells contained 15 N. These cells were then rinsed and put into a media containing only the more common, lighter isotope of nitrogen ( 14 N). As the cells grew and divided in this fresh media, all newly synthesized DNA would contain only the lighter nitrogen isotope, while DNA from the original cells would still contain 15 N. In this illustration above, 15 N labeled DNA is shown in orange and 14 N labeled in green.

The 15 N and 14 N labeled DNA was then tracked using high speed centrifugation and a density * gradient created with cesium chloride (CsCl).

During centrifugation in a CsCl gradient, DNA accumulates in bands along the gradient based on its density. Since 15 N is more dense than 14 N, 15 N enriched DNA accumulates lower down in the centrifuge tube than the 14 N DNA. DNA containing a mixture of 15 N and 14 N ends up in an intermediate position between the two extremes.

By spinning DNA extracted at different times during the experiment, Meselson and Stahl were able to see how new and old DNA interacted during each round of replication.

The beauty of this experiment was that it allowed them to distinguish between the three different hypothesized replication patterns. The key result occurs at the second generation when all three proposed replication patterns give different results in the CsCl gradient.

That Meselson and Stahl's experiment showed the pattern predicted by the semiconservative hypothesis provided the definitive experimental evidence in support of the process proposed by Watson and Crick.

Related Content

• Complementary Nucleotide Bases
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• Complementary nucleotide bases concept questions
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• v.101(52); 2004 Dec 28

Meselson and Stahl: The art of DNA replication

In 2003, the scientific community celebrated the 50th anniversary of James Watson and Francis Crick's landmark 1953 paper on the structure of DNA ( 1 ). The double helix, whose form has become the icon of biological research, was not an instant hit however. The model did not gain wide acceptance until the publication of another paper 5 years later.

Matthew Meselson and Franklin Stahl's experiments on the replication of DNA, published in PNAS in 1958 ( 2 ), helped cement the concept of the double helix. Meselson, a graduate student, and Stahl, a postdoctoral researcher, both at the California Institute of Technology (Pasadena), gave validity to a model that many scientists saw as speculation: how two intertwined and tangled strands of a helix could physically code for the material of inheritance. The Perspective by Philip Hanawalt of Stanford University (Stanford, CA), in this issue of PNAS ( 3 ), reviews the scientific Revolution of this crowning achievement and outlines its subsequent impact on four decades of DNA replication, recombination, and repair research. The two men behind the laborious steps in discovering the semiconservative replication of DNA credit much of their success to timing, hard work, and serendipity.

A Partnership Begins

During his third year of graduate school at the University of Rochester (Rochester, NY), one of Stahl's advisors suggested that he take a physiology course and sent him to the Marine Biological Laboratory in Woods Hole, MA. “I partied my way through that course,” Stahl confesses. “During the partying, I met Meselson,” who was also temporarily at Woods Hole, working as a teaching assistant. During that summer of 1954, the double helix model had been well received but was only truly accepted by an enthusiastic minority of scientists. “Matt had the idea that one ought to be able to use density labels to test Watson's hypothesis,” said Stahl. Although at Woods Hole, Meselson was a graduate student with Linus Pauling at the time at Caltech. There, Meselson had heard Jacques Monod speak about the nature of chemical bonds and enzyme synthesis, which gave Meselson a new technique idea for working with β-galactosidase in bacterial protein synthesis and measuring changes in protein density.

To explore the project, Pauling, whose work centered on x-ray crystallography, sent Meselson to another Caltech professor, Max Delbrück, to learn about the biological aspects of the necessary experiments. Meselson credits Delbrück with giving him the information that would change the nature of the project. As he thrust the Watson and Crick papers toward the young scientist, “He said, `Read these and don't come back until you have,”' Meselson recalls. Up to that point, Meselson admits that he had not been aware of Watson and Crick's work or their DNA structure model.

Stahl planned to go to Caltech for his postdoctoral work, and at Woods Hole he and Meselson decided to collaborate on the density label project in their spare time. “Caltech is a cozy community. It's ruled by ideas, not by walls,” says Stahl. When he arrived at Caltech, Stahl began a bacteriophage project that did not end well after he inadvertently switched the labels on some culture plates. “In the midst of this gloom and doom, Matt came in,” Stahl says. Meselson had finished his main research project and was ready to tackle Watson and Crick's hypothesis. Thus, Stahl changed his focus from bacteriophages to DNA replication.

Not as Simple as It Seems

Meselson and Stahl faced a tangled problem. The Watson and Crick double helix seemed to suggest that the two strands dissociated, each giving rise to a new, complementary strand. The two daughter molecules would thus contain one strand each from the parent molecule, in a semiconservative replication fashion. If replication were conservative, however, the intertwined strands would be replicated as a whole. This would produce one daughter molecule with all original information and one with all new information. The third model, termed dispersive replication, considered that each strand of the daughter molecule could consist of DNA that had been shuffled around so each strand was a hybrid of old and new.

According to Meselson, “There were 2 years of things that didn't work” followed by a year of successful experiments. Jan Drake, then a postdoctoral researcher at Woods Hole, reflected on the years he shared a rented house with Meselson and Stahl and recalls that they all worked the same hard hours kept by many graduate students and postdoctoral researchers today. They would often discuss their work over dinner before returning to the laboratory in the evening. Despite the long hours, results were not immediately forthcoming. Yet perseverance prevailed, and Meselson and Stahl finally designed a successful experiment that would help distinguish new daughter strands from the parent strand.

Hanawalt's Perspective ( 3 ) outlines the intricacies of the differential nitrogen ( 14 N and 15 N) labeling and subsequent separation of the DNA. The experiments demonstrated that Watson and Crick's model of the double helix could replicate itself in a concerted, semiconservative fashion, and the results were published in PNAS after being communicated by Delbrück.

The Legacy of Elegant Peaks

Now, more than 45 years later, the paper is still held aloft for its clarity. Looking back, though, Meselson says the paper has “one thing I wish weren't there.” At the time, published research from an established scientist, Paul Doty ( 4 ), seemed to show that salmon sperm DNA did not come apart when heated. Meselson and Stahl's research could then have two implications: either Doty was incorrect or Escherichia coli DNA actually had four strands. Hence, Meselson and Stahl were cautious with their wording and used the term “subunit” instead of “strand.” “We were little graduate students,” Meselson says. He and Stahl were wary of contradicting an established scientist. “I wish we had had the courage. You should believe in your convictions,” says Meselson. Doty's conclusions were later found to be incorrect because the instruments used were not sensitive enough to detect the DNA molecular weight changes.

Stahl credits the beauty and success of their paper to two things. First, the “delightfully clean data” were serendipitous. The clean data peaks they observed resulted from the DNA fragmenting during handling; unfragmented DNA would not have separated as nicely. Stahl likens pipetting DNA to “throwing spaghetti over Niagara Falls.” The stress of the pipetting caused tremendous shearing of the DNA, although they did not realize this at the time, nor did they realize how critical this would be to obtaining clean peaks. In addition, Meselson was a “stickler for clarity,” said Stahl. “Every single word in that paper was discussed several times before being allowed to keep its position in the sentence.”

Such clean data and clear writing, in addition to the significance of the paper for the field of molecular biology, have placed Meselson and Stahl's experiment on the pages of many a syllabus. At the Massachusetts Institute of Technology (Cambridge, MA), Professor of Biology Tania Baker says the experiment is part of a course required of all molecular biology graduate students. “It is a very nice test of a model of replication,” she says. “Conceptually, it's a very important technique.”

Today, the “little graduate students” stay in touch. Stahl is a professor at the University of Oregon (Eugene, OR), and Meselson is a professor at Harvard University (Cambridge, MA). As definitive as the 1958 paper may appear in its elegance and simplicity, its greater legacy is the subsequent research it has fostered. Cold Spring Harbor Laboratory (Cold Spring Harbor, NY) hosts a meeting for scientists in the field of DNA replication every other year. President and CEO Bruce Stillman acknowledges that it is not a large field—the attendees can fit into a single auditorium—but states that it is a very active one. Stillman says, “Forty-five years after Meselson and Stahl, we've still got work to do.”

• Biology Article
• Dna Replication Experiment

DNA Replication and Meselson And Stahl's Experiment

Literally, replication means the process of duplication. In molecular biology, DNA replication is the primary stage of inheritance. Central dogma explains how the DNA makes its own copies through DNA replication, which then codes for the RNA in transcription and further, RNA codes for the proteins by the translation.

Let’s go through Meselson and Stahl Experiment and DNA replication.

Meselson and Stahl Experiment

Meselson and Stahl Experiment was an experimental proof for semiconservative DNA replication. In 1958, Matthew Meselson and Franklin Stahl conducted an experiment on E.coli which divides in 20 minutes, to study the replication of DNA.

Semi conservative DNA Replication through Meselson and Stahl’s Experiment

15 N (heavy) and 14 N (normal) are two isotopes of nitrogen, which can be distinguished based on their densities by centrifugation in Ca,esium chloride (CsCl). Meselson and Stahl cultured E.coli in a medium constituting 15 NH 4 Cl over many generations. As a result, 15 N was integrated into the bacterial DNA. Later, they revised the 15 NH 4 Cl medium to normal 14 NH 4 Cl. At a regular interval of time, they took the sample and checked for the density of DNA.

Observation

Sample no. 1 (after 20 minutes): The sample had bacterial DNA with an intermediate density. Sample no. 2 (after 40 minutes): The sample contained DNA with both intermediate and light densities in the same proportion.

Based on observations and experimental results, Meselson and Stahl concluded that DNA molecules can replicate semi-conservatively. Investigation of semi-conservative nature of replication of DNA or the copying of the  cells , DNA didn’t end there. Followed by Meselson and Stahl experiment, Taylor and colleagues conducted another experiment on Vicia faba (fava beans) which again proved that replication of DNA is semi-conservative.

Also Read:  DNA Structure

DNA Replication

DNA is the genetic material in the majority of the organisms.  Structurally, it is a double-stranded helical structure which can replicate.

DNA replication is the process by which the DNA makes multiple copies of itself. It was originally proposed by Watson and Crick. DNA replication proceeds as follows:

• Primarily during this process, two DNA strands will open and separate.
• As the strands are separated, the enzymes start synthesizing the complementary sequence in each of the strands. That is, each parental strand will act as a template for the newly synthesized daughter strands.

Since the new DNA strands thus formed have one strand of the parent DNA and the other is newly synthesized, the process is called semiconservative DNA replication.

DNA Replication Fork

Also Read:  DNA Replication

Frequently Asked Questions

Which mode of replication did the messelson and stahl’s experiment support.

Messelson and Stahl’s experiment supported the semi-conservative mode of replication. The DNA was first replicated in 14N medium which produced a band of 14N and 15N hybrid DNA. This eliminated the conservative mode of replication.

What are the different modes of replication of DNA?

The different modes of replication of DNA are:

• Semiconservative
• Conservative

How are semi-conservative and conservative modes of replication different?

Semi-conservative mode of replication produces two copies, each containing one original strand and one new strand. On the contrary, conservative replication produces two new strands and would leave two original template DNA strands in a double helix.

What is the result of DNA replication?

The result of DNA replication is one original strand and one new strand of nucleotides.

What happens if DNA replication goes wrong?

If DNA replication goes wrong, a mutation occurs. However, if any mismatch happens, it can be corrected during proofreading by DNA Polymerase.

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Meselson and Stahl Experiment

Meselson and Stahl experiment gave the experimental evidence of DNA replication to be semi-conservative type . It was introduced by the Matthew Meselson and Franklin Stahl in the year 1958 . Matthew Meselson and Franklin Stahl have used E.coli as the “ Model organism ” to explain the semiconservative mode of replication.

There are three modes of replication introduced during the 1950s like conservative, semi-conservative and dispersive. The researchers were confused between these three that what could be the actual pattern of DNA replication. In 1958, Matthew Meselson and Franklin Stahl presented their research, where they concluded that the replication of DNA is semiconservative type .

Matthew Meselson and Franklin Stahl have conducted several experiments after the discovery of DNA structure (by the two scientists Watson and Crick ). Watson and Crick’s model is widely accepted to demonstrate the replicative model of DNA. We will discuss the definition, steps and observation of the Meselson and Stahl experiment along with the semi-conservative model of DNA.

Content: Meselson and Stahl Experiment

Semi conservation model of dna, meselson and stahl experiment steps, observation, definition of meselson and stahl experiment.

Meselson and Stahl Experiment gave us the theory of semi-conservative replication of DNA. They have taken E.coli as the model organism and two different isotopes, N-15 and N-14 . The N-15 is the heavier isotope, whereas N-14 is the lighter or common isotope of nitrogen. Meselson and Stahl performed their experiment by first growing the E.coli in the medium containing 15 NH 4 Cl for several generations. They observed that the heavy isotope has incorporated in the genome of E.coli and the cells become more substantial due to 15 N heavy isotope.

Meselson and Stahl then transferred the E.coli cells incorporated with 15 N isotope to the medium containing 14 NH 4 Cl for several generations. After every twenty minutes, the E.coli cells multiply. For the processing of DNA, the cells were centrifuged by the addition of Caesium chloride, resulting  in the formation of the concentration gradient. As a result, light, intermediate and heavy DNA strands will get separated.

After completing their experiment, Meselson and Stahl concluded that after each cell division, half of the DNA would be conserved for every next generation. Therefore, this experiment proves that the DNA replication obeys the semi-conservative mode of replication in which 50% of the DNA conserve for every next generation in a way like 100%, 50%, 25%, and 12.5% and so on.

It is the type of DNA replication. The term semi means “ Half ” and conservative means “ To store ”. The semi-conservative DNA replication results in the two daughter DNAs after the parent DNA replication.

In the two daughter DNA’s, each strand will contain a mixture of the parent DNA’s template strand, and the other with a newly synthesized strand (in F-1 gen ). When the parental DNA replicates, half of the 100%, i.e. 50% of the DNA is conserved by having parent strand and the remaining 50% will produce newly synthesized strands.

After the F-1 gen, the multiplication of the cell will get double, which will produce four DNA strands (in F-2 gen ). In F-2 gen half of 50%, i.e. only 25% of DNA is conserved by having parental strands, and the remaining 75% will produce newly synthesized strands.

• Growth of E.coli : First, the E.coli were grown in the medium containing 15 NH 4 Cl for several generations. NH 4 provides the nitrogen as well as a protein source for the growth of the E.coli. Here, the 15 N is the heavy isotope of nitrogen.
• Incorporation of 15 N : After several generations of E.coli, Meselson and Stahl observed that the 15 N heavy isotope has incorporated between the DNA nucleotides in E.coli.
• Transfer of E.coli cells : The DNA of E.coli labelled with 15 N isotope were transferred to the medium containing 14 NH 4 Cl . Here, the 14 N is the light isotope of nitrogen. The E.coli cells were again allowed to multiply for several generations. The E.coli cells will multiply every 20 minutes for several generations.
• Processing of DNA : For the processing or separation of DNA, the E.coli cells were transferred to the Eppendorf tubes. After that, caesium chloride is added, having a density of 1.71 g/cm 3 (the same of DNA). Finally, the tubes were subjected to high-speed centrifugation 140,000 X g for 20 hours.

The result, after two generations of E.coli, the following results were obtained:

In the F-1 generation : According to the actual observations, two DNA strands (with a mixture of both 15 N and 14 N isotopes) will produce in F-1 gen. The above diagram shows that the semiconservative and dispersive model obeys the pattern of growth explained by Meselson and Stahl.

Thus, it is clear that the DNA does not replicate via “Conservative mode”. According to the conservative model, the DNA replicates to produce one newly synthesized DNA and one parental DNA. Therefore, the conservative model was disapproved, as it does not produce hybrid DNA in the F-1 generation.

In the F-2 generation : According to the actual observation, four DNA strands ( two with hybrid and the remaining two with light DNA ) will produce in the F-2 generation. The hybrid DNA includes a mixture of 15 N and 14 N. The light DNA strands contain a pure 14 N. The diagram shows that only semi-conservative type of replication gave similar results conducted by Meselson and Stahl. Thus, both the conservative and dispersive modes of replication were disapproved.

Therefore, we can conclude that the type of replication in DNA is “ Semi conservative ”. The offsprings have a hybrid DNA containing a mixture of both template and newly synthesized DNA in the semi-conservative model. After each multiplication, the number of offspring will double, and half of the parental DNA will be conserved for the next generation.

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The Most Beautiful Experiment: Meselson and Stahl

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00:00:19:21 John Cairns said on the telephone in a very excited voice, 00:00:22:24 he had just read Mendel's papers 00:00:27:04 [Imitating Cairns] "and you know they are the most beautiful 00:00:29:22 experiments in Biology." 00:00:32:04 And I gasped and I said, "John, John, you can't say that." 00:00:37:10 You said the Meselson-Stahl experiment 00:00:40:18 was the most beautiful experiment in Biology. 00:00:44:02 [Imitating Cairns] "Oh, did I? 00:00:46:12 Well, I was wrong." 00:00:53:22 Watson and Crick didn't make a discovery. 00:00:56:15 They proposed a model. 00:00:59:05 There are those who believed this model must be true 00:01:01:21 because it was so beautiful. 00:01:04:02 And there were those who believed it must be wrong 00:01:06:18 because biology is complicated. 00:01:09:02 And this model is too simple to be right. 00:01:12:01 Would you say? 00:01:13:05 Exactly, yes, 00:01:14:14 But there was no experimental proof of it. 00:01:17:01 They had a model which made a distinct prediction 00:01:20:18 about how DNA replicates and it needed to be tested. 00:01:24:11 And it's fun to test the hypothesis. 00:01:29:03 We agreed that we were going to work together 00:01:32:08 to figure out whether or not it was right. 00:01:38:23 When Frank and I showed semi-conservative replication, 00:01:43:03 It wasn't just a model, it was something real like that. 00:01:47:08 After our experiment, 00:01:48:18 it was now widely accepted that their model, 00:01:52:06 Watson-Crick model is right. 00:01:54:19 So it became the building block. 00:01:58:00 You might say for all of biology. 00:02:00:00 Yeah. 00:02:04:05 From very early childhood, I mean, practically infancy. 00:02:07:21 I loved science. 00:02:08:22 I loved to put wires together to make little radios, 00:02:13:24 which I could put under my pillow. 00:02:15:11 So my parents wouldn't know that I was listening 00:02:17:24 to them all night long. 00:02:19:21 And then I was very interested to know what makes life work. 00:02:24:08 And there had been, I think, in the house, 00:02:29:22 maybe even in my bedroom, that painting by Michelangelo, 00:02:36:02 God up high and Adam below and they're touching fingers. 00:02:42:11 I don't know if there is a spark. 00:02:45:08 I don't think there's a spark in the picture, 00:02:47:04 but I'm not sure. 00:02:48:15 But to me that meant that life is somehow electrical. 00:02:53:10 God is providing life through a spark. 00:02:57:14 And for some reason that made me interested in 00:02:59:12 electrochemistry. 00:03:01:19 Unlike Matthew, 00:03:02:20 I had no particularly strong interest in science as a youth. 00:03:09:11 What was understood that when I graduated from high school, 00:03:14:02 I would apply to the Naval Academy. 00:03:17:01 That's what my mother had in mind because she thought 00:03:19:09 I would look good in dress whites. 00:03:22:08 But then of course, World War II broke out. 00:03:25:01 So that plan changed fast and she decided 00:03:28:13 I should just go to college. 00:03:30:19 I think I was just too young to understand 00:03:33:13 the courses in humanities. 00:03:35:03 I hadn't had enough life experience to get a grip 00:03:38:23 on the questions they were even thinking about. 00:03:41:23 Science on the other hand was concrete. 00:03:44:17 Children can grasp science and among the sciences, 00:03:50:05 biology was the most appealing. 00:03:52:09 There, the fun was that you could figure out puzzles. 00:03:56:24 That is, there was a rational, concrete, 00:03:59:09 quantitative explanation for what you saw. 00:04:03:03 You could reason backwards as to what must be going on. 00:04:07:10 And that intrigued me enough to know that genetics 00:04:10:14 was something perhaps I could do. 00:04:20:19 I had the great, good luck to become Linus Pauling's 00:04:23:21 last graduate student. 00:04:25:09 His daughter was having a party at their swimming pool 00:04:28:12 and I'm in the water. 00:04:29:09 And Pauling comes out, the world's greatest chemist. 00:04:32:16 I'm all naked, practically, in a bathing suit. 00:04:36:13 And he's all dressed up with a jacket and a vest 00:04:40:04 and a neck tie. 00:04:41:11 And he looked down at me, 00:04:42:09 "Well, Matt, what are you gonna do next year?" 00:04:45:00 And I had already signed up 00:04:46:02 to go to the committee on mathematical biophysics 00:04:52:02 and Linus just looked down at me and he said, 00:04:54:00 "But Matt, that's a lot of baloney. 00:04:56:04 Come be my graduate student." 00:04:58:13 And so if I hadn't taken his course 00:05:00:19 on the nature of the chemical bond, 00:05:03:01 I would have had a very different life. 00:05:04:14 I wouldn't have met Frank, 00:05:06:19 I wouldn't be sitting here, that's for sure. 00:05:10:02 At the end of my PhD exam, 00:05:12:12 as we were walking out of the little exam room, 00:05:14:10 Linus Pauling turned to me and he said, 00:05:16:03 "Matt, you're very lucky you're entering this field 00:05:18:13 just at the right moment." 00:05:19:19 Yeah. 00:05:20:15 At the very beginning. 00:05:22:20 (upbeat music) 00:05:28:13 The first year of my being a graduate student at Caltech, 00:05:31:19 I wanted to get into biology. 00:05:33:17 I was a chemist and I thought the way to do that 00:05:36:12 would be to study molecular structure. 00:05:39:16 The only person who was looking at biology 00:05:42:01 from that point of view, other than Linus Pauling himself 00:05:45:10 was Max Delbruck. 00:05:47:03 He had a fearsome reputation. 00:05:49:13 Nevertheless, I got up my courage and went to see him. 00:05:52:10 He's not a fearsome creature at all really. 00:05:54:11 And the first thing he said was, 00:05:56:15 what do you think about these two papers 00:05:58:12 from Watson and Crick? 00:06:00:11 I said, I'd never heard of them. 00:06:03:01 I was still in the dark ages, 00:06:05:22 and he yelled at me. 00:06:07:08 He said, "Get out 00:06:08:04 and don't come back till you've read them." 00:06:13:12 There were two separate ideas that came together. 00:06:16:07 Crick's idea about how the base pairs linked onto the chains 00:06:21:22 and Jim's idea about how the base pairs were structured. 00:06:26:16 So there are four different building blocks in DNA, 00:06:29:07 adenine, thymine, guanine, and cytosine. 00:06:31:19 The surfaces of the G and the C are complementary 00:06:35:13 to each other and of the A and T are complementary 00:06:39:08 to each other so that they can fit together. 00:06:43:05 The way fingers would fit into a glove. 00:06:46:04 And importantly, when they put G opposite C, 00:06:50:21 the distance of the outside was exactly the same 00:06:55:07 as if they'd put A opposite T. 00:06:58:09 No other combination would give such a regular structure. 00:07:02:03 It was a gorgeous insight. 00:07:05:21 And then from that, 00:07:07:01 they made a hypothesis about how DNA is replicated. 00:07:11:02 It involved the two chains coming apart 00:07:15:12 and each one acting as a template for the synthesis 00:07:20:07 of a new chain on its surface. 00:07:24:12 When it's all done, here we have the two old chains, 00:07:27:24 each one now associated with a brand new chain. 00:07:32:08 What Watson and Crick proposed 00:07:34:07 was enormous stimulus to experimentation. 00:07:37:24 It was irresistibly beautiful. 00:07:39:21 Irresistibly beautiful. 00:07:42:22 Jim Watson was at Caltech the year after 00:07:46:19 he and Francis published their papers. 00:07:49:24 And so I got a chance to talk a lot with Jim then, 00:07:53:12 and that coming summer he was going to go 00:07:56:00 and teach the physiology course at Woods Hole. 00:08:01:16 I was a graduate student at Rochester at the time. 00:08:05:06 My chairman of the department who was also on my committee, 00:08:09:05 said I had to take a course in physiology. 00:08:12:18 And I said, the physiologist teacher here is a jerk. 00:08:15:13 I'll be damned if I'll take his course. 00:08:17:19 Well, send him to Woods Hole 00:08:19:23 to take the physiology course there. 00:08:22:16 And by serendipity, Jim Watson happened to be there 00:08:25:19 with some kid named Meselson hanging along with him. 00:08:29:19 We found that we had in fact deep, common interests. 00:08:33:22 I realized this is a guy who's really very smart 00:08:36:18 and I can learn a lot from him. 00:08:38:15 I remember a haze of beach parties, 00:08:42:03 lectures that I slept through 00:08:45:08 Well it was a kind of paradise. 00:08:48:04 The most interesting people in molecular biology. 00:08:51:08 Most of them were there. 00:08:52:22 So that's how we met. 00:08:54:10 And then it turns out Frank is coming that very September 00:08:57:07 to Caltech. 00:08:58:14 It would be a year from then I would come. 00:09:00:06 Are you sure? 00:09:01:03 Yep. 00:09:02:07 I still hadn't finished my thesis- 00:09:03:03 So I had to wait for a whole year before I saw you again? 00:09:05:18 That's right. 00:09:06:14 He said, when you get to Caltech we'll test Jim's idea. 00:09:11:13 What do you think about testing Jim's idea 00:09:13:22 of how DNA replicates? 00:09:15:19 And then he explained that to me, 00:09:17:21 I'd already heard about it and he explained it to me 00:09:20:23 and I absolutely - I committed, totally. 00:09:24:11 And then when Frank finally got there 00:09:26:22 and I wanted to start right away, he forbade it. 00:09:30:17 Why? 00:09:31:14 He said it would be bad for my character 00:09:34:06 to not complete my x-ray crystallography 00:09:37:09 before starting something new. 00:09:40:17 This tells you a lot about Frank's character. 00:09:46:18 With the Watson and Crick model, 00:09:48:16 the underlying question of course was, 00:09:50:23 was that really the right mechanism? 00:09:53:00 The famous Max Delbruck said no, no, no, no, 00:09:56:19 that model can't be right. 00:09:57:19 And he proposed a different model. 00:10:00:03 As Delbruck put it forth, 00:10:02:01 breaks are introduced in the parental molecule 00:10:05:14 as it's being replicated 00:10:07:13 and then carefully sealed up in certain ways. 00:10:10:16 Others proposed one in which 00:10:12:12 the original DNA molecule stays intact. 00:10:16:01 And the new DNA molecule is made of all new DNA. 00:10:21:14 So there were three targets out there 00:10:24:18 that in principle could be distinguished, 00:10:27:00 if you could trace the fate of the old chains, 00:10:30:11 what becomes of the two old chains. 00:10:32:16 And one step led to the next, really. 00:10:35:09 I mean, the first idea was using density somehow, 00:10:39:10 which is not a very good idea yet, 00:10:41:07 except it leads you to the next one. 00:10:43:03 Matt's idea from the very beginning 00:10:45:10 was that somehow stable isotopes could be used. 00:10:50:01 That would be incorporated into the DNA 00:10:53:02 and impart upon the DNA, a different density. 00:10:56:10 You grow bacteria in a medium, 00:10:59:03 which instead of having this ordinary isotope of nitrogen 00:11:03:11 N14, you can buy nitrogen 15 ammonium chloride, 00:11:09:24 the heavy kind. 00:11:12:11 And if you grow the bacteria for a number of generations, 00:11:15:13 you can be sure that essentially all of the DNA 00:11:19:12 is labeled with heavy nitrogen, good. 00:11:23:08 Now, we resuspend those cells in a medium that just has 00:11:26:13 ordinary, nitrogen 14, the light one. 00:11:30:12 And now the question is as the DNA molecules replicate, 00:11:34:23 how will the heavy nitrogen from those parent molecules 00:11:39:04 be distributed amongst the daughter molecules 00:11:42:19 that are produced in successive duplications? 00:11:46:13 Then some sensitive method for separating DNA, 00:11:50:24 according to its density would be devised. 00:11:54:18 I ran across an article about the centrifugation 00:11:58:06 of cesium chloride solution 00:11:59:24 to measure the molecular weight. 00:12:02:01 If the DNA was in there with the cesium, 00:12:05:17 it would find its position in the density gradient. 00:12:09:07 If it was heavy DNA, 00:12:11:15 it would tend to be down near the bottom of the tube 00:12:14:02 where the cesium was concentrated and the density was high. 00:12:18:14 If the DNA was light DNA, made of light isotopes, 00:12:22:18 it would be higher up in the tube. 00:12:29:00 You could think about it this way. 00:12:30:21 If you jumped into the Great Salt Lake, 00:12:32:21 as we all know you float, 00:12:34:15 you go right to the top because you are less dense 00:12:38:00 than the water. 00:12:38:21 But if you have a bathing suit with pockets in it, 00:12:41:10 and you stuffed some lead weights in your pockets, 00:12:44:22 you'll sink down. 00:12:46:05 Cause you're more dense than the water. 00:12:48:22 Now imagine that the salt in the Great Salt Lake 00:12:51:22 is not uniformly distributed, 00:12:54:09 but is concentrated near the bottom 00:12:57:12 and rather less concentrated near the top. 00:13:01:12 Now, if you put just the right number of heavy weights 00:13:04:13 in your pocket, you won't float because you'll be too dense. 00:13:09:05 You won't float at the top and you won't go all the way 00:13:11:13 to the bottom because you're not dense enough. 00:13:14:03 You'll instead come to rest somewhere, 00:13:16:13 halfway between the top and the bottom, 00:13:19:01 you will have found your place in that gradient. 00:13:23:17 And that's the very basis by which the experiment 00:13:26:12 finally worked and worked so beautifully. 00:13:29:01 And then it was just a question of looking 00:13:30:22 in the centrifuge while it's running. 00:13:33:24 And when it reaches equilibrium to see where 00:13:37:07 the heavy and light DNA are. 00:13:39:02 All the makings were there, 00:13:40:13 then to do the experiment itself, 00:13:42:21 it was obvious that the experiment was going 00:13:45:08 to give an answer. 00:13:46:23 Driving it all was the fact that Frank 00:13:49:14 wanted to know how life works. 00:13:57:12 Yeah, yeah. 00:14:01:14 [Mumbles] 00:14:04:09 I don't know that drove it all but- 00:14:07:14 Each person is trying to come up with something 00:14:09:21 as a gift to the other guy. 00:14:12:02 That's true. 00:14:12:23 I think 00:14:13:19 That's true 00:14:14:15 So it becomes a very connected 00:14:17:02 relationship because the next day you want 00:14:20:12 to have something to offer. 00:14:23:09 Matt was ready to step out into an area, 00:14:26:22 pretty heavily uncharted, 00:14:29:23 to answer an important question. 00:14:32:08 And the pieces had to be built as he went along. 00:14:36:15 (upbeat music) 00:14:40:18 The prediction of the Watson and Crick model, 00:14:43:04 was the two parent chains come apart. 00:14:45:00 Each one makes a new daughter molecule 00:14:47:01 and that's replication. 00:14:48:16 So that would predict that after exactly one generation, 00:14:52:16 when everything has doubled in the bacterial culture, 00:14:56:02 that you'd find the DNA molecules all have one old strand, 00:15:01:12 which is labeled heavy. 00:15:03:01 And one new strand, 00:15:05:01 which is labeled light and therefore their density 00:15:08:01 should be halfway between fully heavy and fully light, 00:15:12:06 that would be the prediction for what you see 00:15:15:07 at exactly one generation. 00:15:17:08 What do you predict to see for the next generation? 00:15:20:17 Well, each molecule would, again, separate its chains. 00:15:24:08 One of which is heavy. 00:15:26:00 The other of which is light and the only 00:15:28:14 growth medium available is light growth medium. 00:15:32:08 Then the light chain would make another light chain 00:15:35:04 to go with it, a complement. 00:15:37:05 The heavy chain would make another, 00:15:39:15 a light chain to go with it. 00:15:42:00 So after two generations you have DNA, 00:15:45:11 half of which is half heavy. 00:15:47:13 And the other half of which is all light 00:15:53:13 And fantastically, 00:15:55:05 that's exactly the result that one could see. 00:16:02:24 In order to say that the Watson-Crick model 00:16:06:08 fits the data very well, but the other two models do not, 00:16:10:14 we have to see what they'd predict. 00:16:12:23 Start with the Dispersive Model. 00:16:15:10 After one generation, 00:16:17:02 the two molecules resulting would indeed be half heavy, 00:16:21:10 but in the next generation, 00:16:23:10 there would be a subsequent dispersion of the label. 00:16:26:18 So you'd be getting molecules that were 00:16:29:20 three quarters light, and one quarter heavy. 00:16:34:22 And in each generation, 00:16:36:10 the molecules would get lighter and lighter. 00:16:39:16 The fully Conservative Model simply imagined that duplex DNA 00:16:44:16 fully heavy now, somehow created the appearance of a fully 00:16:51:09 light duplex molecule in which both chains 00:16:54:07 are made of light DNA. 00:16:59:15 Most of the times when you get an experimental result, 00:17:03:19 it doesn't speak to you with such clarity. 00:17:07:19 These pictures of the DNA bands interpreted themselves. 00:17:18:14 It felt like a...supernatural. 00:17:21:22 It felt like you were in touch with the gods 00:17:24:10 or something like that. 00:17:25:16 I remember I presented this result that summer 00:17:29:16 early in the summer in France at a phage meeting, 00:17:33:23 complete with the photographs 00:17:36:02 of the density gradient bandings. 00:17:39:05 And at the end of it, 00:17:41:17 I stopped and there was total silence and somebody said, 00:17:45:16 "Well, that's it." 00:17:53:21 The intellectual freedom at Caltech. 00:17:56:02 We could do whatever we wanted. 00:17:58:00 It was very unusual for such young guys 00:18:00:19 to do such an important experiment. 00:18:02:23 So suddenly, whereas before that, 00:18:05:12 like Max would be talking with Sinsheimer 00:18:08:00 about the genetic code. 00:18:09:23 And before we did our experiment, 00:18:11:10 I was definitely not - at least 00:18:13:11 I felt I wasn't - supposed to be at those discussions. 00:18:16:15 But afterwards, I could be a full member. 00:18:20:06 We had this wonderful house, 00:18:21:15 big house across the street from the lab. And our roommates, 00:18:26:19 we all, 00:18:27:15 we talked about these experiments at almost every dinner. 00:18:31:01 So we had this wonderful intellectual atmosphere, 00:18:35:11 John Drake, Howard Temin. 00:18:38:00 Why are you frowning? 00:18:39:06 He told the dirtiest jokes I've ever heard. 00:18:41:04 No that was Roger Milkman. 00:18:42:20 [Crosstalk] 00:18:45:11 Positions one and two. 00:18:46:18 That's true, that's true, that's true. 00:18:49:05 So it was a very lively, intense, friendly atmosphere. 00:18:56:12 It was lively enough and conveniently located enough 00:19:00:12 that over time we had visits from William O. Douglas, 00:19:05:17 Judge Douglas. 00:19:06:13 Judge Douglas of the Supreme Court 00:19:08:11 And here Dick Feynman probably one of the world's greatest 00:19:13:13 physicists at that time, 00:19:15:04 or maybe ever, palled around with us. 00:19:17:17 He came over to our big house and played his drums, 00:19:22:00 sat down on the floor, played the drums. 00:19:24:21 I'm just a graduate student 00:19:26:03 and he's the world's greatest physicist, 00:19:29:17 but that's what it was like. 00:19:30:22 It was a very friendly wide open place. 00:19:33:10 Frank and I are very lucky. 00:19:36:20 The way I think of it is that there's a river, 00:19:39:17 which is a period of time when the fundamental things, 00:19:42:21 the structure of DNA, how replication happens, 00:19:45:10 the genetic code. 00:19:47:06 And then, when these problems are solved. 00:19:50:22 There are lots of little rivulets. 00:19:51:18 The river divides into thousands of branches 00:19:55:16 using these fundamental insights into how life works 00:20:00:16 and applying them to specific questions, 00:20:03:14 questions of disease etc. 00:20:07:09 So to me, with some exceptions, 00:20:11:21 this was a really interesting time 00:20:14:00 when it was still a big river. 00:20:16:04 Also, now you can cut this out, 00:20:19:04 but also the Meselsons, Matt's parents, were kind enough to 00:20:22:14 keep the liquor cabinet fully stocked at all times. 00:20:29:22 (upbeat music) 00:20:49:15 My throat is a little bit? 00:20:51:22 I have a cough drop 00:20:54:06 (whispering) I don't want a cough drop. I want a non-alcoholic beer. 00:20:58:21 I require a margarita. 00:21:00:22 I've worked for the CIA. 00:21:04:08 I vaporized many people, including many of your friends, 00:21:08:16 Big black beard, 00:21:09:19 and blew out some of his pipe smoke and still 00:21:12:20 holding his pipe stem in his teeth said, 00:21:15:01 "Oh Matt history is just what people think it was."

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Talk Overview

Matt Meselson and Frank Stahl were in their mid-20s when they performed what is now recognized as one of the most beautiful experiments in modern biology. In this short film, Matt and Frank share how they devised the groundbreaking experiment that proved semiconservative DNA replication, what it was like to see the results for the first time, and how it felt to be at the forefront of molecular biology research in the 1950s. This film celebrates a lifelong friendship, a shared love of science, and the serendipity that can lead to foundational discoveries about the living world.

Please head to the Science Communication Lab’s website for more films like this along with educator resources, full video transcript, and most up to date content.

Speaker Bio

Frank stahl.

Frank Stahl received his PhD at the University of Rochester, where he studied genetic recombination in phage. He performed postdoctoral studies at Caltech, during which he completed the famous Meselson-Stahl experiment, and joined the faculty at the University of Oregon in Eugene in 1959. He is now an emeritus faculty member who enjoys teaching and… Continue Reading

Matthew Meselson

Dr. Meselson has made important contributions to the areas of DNA replication, repair and recombination as well as isolating the first restriction enzyme. Currently, he is Professor of Molecular and Cellular Biology at Harvard University, where his lab studies aging in the model organism bdelloid rotifers. Meselson is also a long-time advocate for the abolition… Continue Reading

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Related Resources

Meselson M. and Stahl F. The replication of DNA in Escherichia coli . PNAS July 15, 1958 44 (7) 671-682.

Teaching resources from XBio: How DNA Replicates

Sarah Goodwin (Wonder Collaborative): Executive Producer Elliot Kirschner (Wonder Collaborative): Executive Producer Shannon Behrman (iBiology): Executive Producer Brittany Anderton (iBiology): Producer Derek Reich (ZooPrax Productions): Videographer Eric Kornblum (iBiology): Videographer Rebecca Ellsworth (The Edit Center): Editor Adam Bolt (The Edit Center): Editor Gb Kim (Explorer’s Guide to Biology): Illustrations Chris George: Design and Graphics Maggie Hubbard: Design and Graphics Marcus Bagala: Original music Samuel Bagala: Original music

Reader Interactions

ANGELA DIXON says

February 8, 2021 at 9:39 pm

Thank you – I cannot tell you how much I enjoyed this video. It was as if I was sitting in Dr. Stahl’s living room, having a conversation with these two great scientists. What an elegant experiment! You have really captured the essence of two incredible scientists in this video.

Marieke Mackintosh says

March 23, 2021 at 12:08 pm

Thank you for sharing this incredible footage of these brilliant human beings. What a joy it is to watch them reminisce and teach. I cannot wait to show this to my students.

Neeraja Sankaran says

August 30, 2022 at 7:00 am

Hi.. this is not a comment except to say that this is a beautiful video. Could you give me the full citation please, I’d like to include it in a bibliography

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The Meselson-Stahl Experiment: “the Most Beautiful Experiment in Biology”

• First Online: 24 November 2020

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• Allan Franklin 3 &
• Ronald Laymon 4  

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In this chapter we will examine the Meselson-Stahl experiment which decided the issue between two models of DNA replication. The experimental result was held not to require replication. A second experiment of a rather different nature was used to eliminate a third possibility, and whereby contrast that experiment was further refined so as to yield a more accurate result. These experiments set the stage for the creation of new competing accounts of DNA replication all of which—given the framework established by the Meselson-Stahl experiments—were eliminated by experimental tests leaving only the Watson-Crick model in play.

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For an excellent history of this complex episode and complete references see Holmes ( 2001 ).

See Delbruck and Stent ( 1957 ) and Holmes ( 2001 , pp. 15–29, 393–394).

Often referred in textbooks as being N 14 labeled as opposed to unlabeled.

It should be noted that the consistent locations of the various bands as a function of their association with the labeling was determined and validated by the calibration procedure of superimposing the data images at generation points 0 and 1.9 as shown in the penultimate data row.

To see how this goes trace the replication paths indicated in Fig.  2.5 and note that at the end of the first generation there are now (as per the first part of the first conclusion) twice as many subunits are there were parental subunits to begin with. And then at the next generation there will again (as per the second part of the first conclusion) be twice as many progeny but where only one half will be half labeled. Thus, the number of half-labeled subunits at the end will be twice the number of parental subunits at the initial stage of the process.

In a later chapter on the discovery of the positron we’ll review another instance where the theoretical results were deduced from the experimental data (augmented by non-controversial theoretical assumptions) when Patrick Blackett made such a derivation in order to clearly demarcate what his experimental results demonstrated from their role in confirming Dirac’s hole theory of the production and nature of the positron.

Younger readers may need to be reminded that email was not always available.

For an extensive analysis of null experiments and the sense in which they satisfy demands for replication see Franklin and Laymon ( 2019 ).

There is, though, a caveat that we need to make. There emerged on the basis of further experimentation and theoretical development a realization that the sharpness of the separation bands reported by Meselson and Stahl was due in part to an unrecognized but uniformly consistent fragmentation of the DNA molecule that occurred during the preparation of the DNA samples. But the defect was not claimed in any way to lend experimental support for conservative replication, and moreover new experiments continued to support semi-conservative replication. For the details see Holmes ( 2001, pp. 395–397 ). As Thomas Kuhn would have described it, these further developments were part of normal science conducted in response to the Meselson and Stahl experiment understood as a paradigm (Kuhn 1962 , pp. 23–42).

There is an argument to be made even if it’s true that dispersive replication is not formally inconsistent with semi-conservative replication, the constraints imposed by semi-conservative replication on dispersive replication are so severe and restrictive that dispersive replication is effectively disconfirmed because of the evident difficulties that would be involved in creating a developed and specifically testable version of dispersive replication. Alternatively stated, once was enough for the Meselson and Stahl experiment to have effectively discouraged any further pursuit of dispersive replication as a promising approach to understanding the replication of DNA. Viewing dispersive replication as having been effectively disconfirmed in this way provides a charitable explanation why many of the textbooks claim that the Meselson and Stahl experiment disconfirmed not only conservative replication but also dispersive replication. See, for example (Lehninger 1975 , pp. 659–61) where it is claimed that the results of the Meselson and Stahl experiment “are exactly those expected from the hypothesis of semiconservative replication proposed by Watson and Crick; whereas they are not consistent with the alternative hypotheses of conservative or dispersive replication.”.

One can make the representation more realistic (in the sense of representing dispersion at multiple locations) by simply stacking the rectangular modules and making them of different height (and internal proportionality) to accommodate different dispersion locations. In which case, the argument for consistency with semi-conservative replication carries through as before.

Meselson and Stahl were here relying on a method of density and weight determination that they had developed along with Jerome Vinograd which was reported in Meselson et al. ( 1957 ).

For a brief review of this development see Holmes ( 2001, 394–395 ).

Cairns, J. 1962. Proof that the replication of DNA involves the separation of the strands. Nature 194: 1274.

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Cairns, J. 1963. The bacterial chromosome and its manner of replication as seen by autoradology. Journal of Molecular Biology 6: 298–213.

Cavalieri, L., and B.H. Rosenberg. 1961. The replication of DNA: III. Changes in the number of strands in E. coli DNA during its reproduction. Biophysical Journal 1: 337–351.

Cavalieri, L., and B.H. Rosenberg. 1962. Nucleic acids: Molecular biology of DNA. Annual Review of Biochemistry 31 (258): 247–270.

Delbruck, M., and G.S. Stent. 1957. On the mechanism of DNA replication. In The chemical basis of heredity , ed. W.D. McElroy and B. Glass. Baltimore: Johns Hopkins University Press.

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Holmes, F.L. 2001. Meselson, Stahl, and the replication of DNA: A history of “The Most Beautiful Experiment in Biology” . New Haven: Yale University Press.

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Lehninger, A.L. 1975. Biochemistry . New York: Worth.

Meselson, M., and F.W. Stahl. 1958. The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences (USA) 44: 671–682.

Meselson, M., F.W. Stahl, et al. 1957. Equilibrium sedimentation of macromolecules in density gradients. Proceedings of the National Academy of Sciences 43 (7): 581–588.

Stryer, L. 1975. Biochemistry . New York: W.Y. Freeman.

Watson, J.D. 1965. Molecular biology of the gene . New York: W.A. Benjamin.

Watson, J.D., and F.H.C. Crick. 1953. Genetical implications of the structure of deoxyribonucleic acid. Nature 171: 964–967.

Watson, J.D., and F.H.C. Crick. 1958. A structure for deoxyribonucleic acid. Nature 171: 737–738.

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Franklin, A., Laymon, R. (2021). The Meselson-Stahl Experiment: “the Most Beautiful Experiment in Biology”. In: Once Can Be Enough. Springer, Cham. https://doi.org/10.1007/978-3-030-62565-8_2

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An Elegant Experiment to Test the Process of DNA Replication: The work of Meselsohn and Stahl

by Nathan H Lents, Ph.D.

Did you know that many classic experiments in science are famous not because of their complexity but because of their simplicity? This is the case with Meselson and Stahl’s classic experiment in DNA replication. In one of the most famous experiments in molecular biology, Meselson and Stahl elegantly tested three scientific hypotheses with one simple design.

Around the world, there are thousands of scientists performing experiments at any given moment. Every once in a while, an experiment is performed and published that appears so clever, so important, and so successful in its goals, that it is destined to be cheered by scientists far and wide and taught in science classrooms for decades to come. However, with the passage of time, these so-called "classic experiments" seem more dramatic, ingenious, and clear than they were in their contemporary timing as the memories of complications, contradictions, and controversy fade. Nevertheless, what often sets apart these key experiments is not that they are especially complex, but that they are elegantly simple. The power of simplicity in an experiment is that it reduces the chance of alternative explanations for the results.

As explained in our module Theories Hypotheses , and Laws , a key feature of the modern scientific method is that valid scientific hypotheses make predictions that can be tested. Thus, the testing of predictions is a major part of scientific research , and part of the historic nature of many classic experiments is that they tested the predictions of a key scientific hypothesis in a way that provided a clear answer. The 1958 experiment by Matthew Meselson and Franklin Stahl is an example of such an experiment, and is one of the most famous in all of molecular biology. With one cleverly designed experiment, they tested the predictions of three different scientific hypotheses simultaneously, and the field of DNA biology was changed forever.

• DNA replication

Following the discovery of DNA as the genetic material (see DNA I ), the new field of molecular biology focused intently on how DNA functions. One of the most important features of DNA is its ability to be copied accurately. When a cell , whether it is a yeast , a bacterium, or a human cell, divides in two, both resulting cells are genetically identical to each other and to the original parent cell. Thus, prior to division, a cell must somehow copy all of its DNA so that both resulting cells have the full complement of genetic material. Indeed, scientists such as Edwin Chargaff and others had observed that the amount of DNA in a cell doubles prior to cell division. The pool of DNA is then split equally between the two daughter cells, so that both have the same amount of DNA as the original parent cell had. But how exactly this DNA doubling takes place was at first a mystery, and scientists began to propose several possible mechanisms, or "models" of DNA replication?

Following the proposal and eventual acceptance of the Watson-Crick model of DNA structure, molecular biologists believed that each strand of DNA somehow served as a copy-template for the synthesis of a new DNA molecule (see our DNA II module for more information). However, conundrums remained. Most importantly, scientists had a difficult time envisioning how two strands of DNA that are immensely long and twisted around each other could separate from each other without resulting in the breakage of the strands or them becoming hopelessly entangled. In addition, scientists wondered how the two strands could be pulled apart given the enormous number of hydrogen bonds holding them together. Some envisioned replication as proceeding in short stretches, while others imagined a continuous process much like a zipper. This paradox even caused some prominent scientists of the day to doubt the double-helix structure of DNA altogether. Nevertheless, scientists began working on possible theoretical solutions to the separation of two intertwined DNA strands and by the late 1950s, three hypothetical models for DNA synthesis were being hotly debated: the conservative model, the semi-conservative model, and the dispersive model. Figure 1 below provides a diagram of each of these mechanisms.

Figure 1: Three competing models of DNA replication. This diagram shows the three competing models of DNA replication in the 1950s and 1960s.

Briefly stated, the conservative model of DNA replication holds that when DNA is replicated prior to cell division, one of the DNA double-strands receives all newly replicated DNA in both strands, while the other receives only the two original DNA strands in the parent cell. The semi-conservative model (also called the "zipper model" by James Watson), however, holds that the original two strands of DNA are split from each other and that the two daughter molecules are each comprised of one "old" strand of DNA from the parent cell, and one newly replicated strand. Finally, the dispersive model holds that the DNA is copied in short stretches and that both daughter DNA strands will receive a mixture of the original parental DNA and newly replicated DNA. Each of these three possible models had been proposed by different scientists and each had certain advantages in explaining the separation of the intertwined parental DNA. However, evidence to disprove or support any of the models was scarce.

Comprehension Checkpoint

• Design of the Meselson and Stahl experiment

This changed when Matthew Meselson and Franklin Stahl, two scientists working at the California Institute of Technology (CalTech), constructed an ingenious experiment that tested all three models at the same time. To understand how this experiment worked, it is important to remember how atomic isotopes behave. Although a heavier isotope of a given atom behaves in a completely normal manner in chemical reactions , the presence of an extra neutron (or more) gives the atom a slightly higher atomic mass . As a result, molecules that contain these "heavy" isotopes are more dense . This small difference in density allows scientists to physically separate molecules with different isotopes based on the differences in their density.

For their experiment , Meselson and Stahl used a special form of nitrogen: 15 N. Normally, almost all of the nitrogen in any given cell is 14 N and thus contains seven neutrons in addition to its seven protons . So, 15 N, with eight neutrons, is considered "heavy nitrogen" (but it is not radioactive). When growing cells are fed heavy nitrogen, the 15 N isotope enters the cells' metabolism and significant amounts of it will be incorporated into the nitrogen-rich nucleotides and DNA . Thus, the DNA of cells grown with 15 N in their food source would be more dense than that of normal cells. The power of having DNA of different densities is that they can be separated by centrifugation .

For this procedure, cells were first broken open; then the cellular contents (called the "crude extract") were mixed with a solution of the heavy salt cesium chloride and placed in a centrifuge cell with clear quartz walls that allowed the solution to be photographed while spinning. The cell was then spun in a centrifuge at very high speeds for many hours and the heavy cesium ions were pulled towards the bottom of the cell by centrifugal force . Eventually, equilibrium was reached and a "density gradient" was established in the cell with the bottom containing the highest concentration of cesium and the top of the tube containing the lowest. Inside a density gradient like this, all the molecules from the cell extract, including the DNA , will "float" or "sink," migrating to the spot in the gradient that corresponds to their density. The most dense molecules will be pulled toward the bottom of the cell, while less dense molecules will settle higher in the cell, as shown in Figure 2.

Figure 2: The principle of density gradient centrifugation. When a liquid solution containing many large protein and DNA components is placed into a test tube or centrifuge cell and spun at high speed over many hours, the individual molecules separate based on their density. The most dense molecules fall to the bottom, the least dense remain at the top.

Before beginning their analysis of DNA replication , Meselson and Stahl first showed that DNA made with regular 14 N could be separated from DNA containing heavy 15 N. They accomplished this by growing two separate batches of Escherichia coli bacteria , feeding each batch a different nitrogen isotope . Then, they broke the bacterial cells open, mixed the extracts from both batches into one centrifuge cell, and spun it to establish the density gradient. To detect the DNA, they shined ultraviolet (UV) light on the spinning centrifuge cell because DNA absorbs UV light and thus casts a shadow during exposure of photographic film. Below, in Figure 3, is a black-and-white image of their data , and you can clearly see two bands of DNA, one lower in the cell and thus, more dense than the other.

Figure 3: Density gradient centrifugation of a mixture of 15 N-DNA and 14 N-DNA. Meselson and Stahl first showed that they can separate a mixture of DNA of the two different densities. The picture on the left is a UV photograph showing the banding of DNA of different densities following centrifugation. The graph on the right is a trace of the intensity of the bands in the picture.

• How the experiment tested all three DNA replication hypotheses

Next, Meselson and Stahl did something interesting. They grew a large batch of bacteria in heavy nitrogen ( 15 N) and then switched the bacteria to a diet that contained only regular nitrogen ( 14 N). This allowed them to distinguish between pre-existing DNA from the parental cells and newly synthesized DNA, because any newly synthesized DNA strands would contain 14 N and be less dense . They used this experimental set up to put the three possible models of DNA replication to the test.

Like all proper scientific hypotheses , the three models of DNA replication each make certain predictions, and testing hypothetical predictions is a key part of scientific research . In the case of Meselson and Stahl's experiment , the predictions that each of these models makes are as follows. If the conservative model of DNA replication is true, then one would predict that the bacterial cells grown for one generation (20 minutes) with 14 N would have two different kinds of DNA: the original DNA would be the density of DNA grown with only 15 N nitrogen, while both strands of the new DNA molecules would be the lighter 14 N DNA band. However, if either the semi-conservative or the dispersive models of DNA replication are correct, the double-stranded DNA inside the bacteria after one generation would be a mixture of old and new DNA, and thus, one strand would be made of 15 N and one of 14 N DNA. Thus, this "hybrid" DNA would be an intermediate density halfway between the 14 N and 15 N bands of DNA. In Figure 4 below, you can see what the three models of DNA replication predict will happen in the Meselson and Stahl experiment, followed by what they actually observed.

Figure 4: Density gradient centrifugation of E. coli DNA after one cell division. Top panel: the three experimental predictions of three competing models of DNA replication. Bottom panel: The actual data. E. coli grown in 15 N DNA were switched to 14 N and then harvested at five different time points. The DNA was centrifuged resulting in the banding pattern shown here.

As you can see from their results above, after one generation of cell division, the total DNA of the growing bacterial cells had an intermediate density , halfway between that of 14 N and 15 N DNA. This strongly disproved the conservative model of DNA, which held that the original two strands of DNA would persist and remain bound to each other and a wholly new copy of two DNA strands would be synthesized. In other words, the conservative model of DNA replication would predict that, after one generation, half of the DNA molecules would have only 15 N DNA and half would have 14 N DNA, which would appear as two distinct bands of DNA density. But as seen above, all DNA molecules were of intermediate density. However, the two remaining models, semi-conservative and dispersive, were still consistent with these results. In order to examine the two remaining models of DNA replication, we must examine additional generations of bacterial growth with the normal 14 N isotope of nitrogen. In Figure 5 below are the predictions of all three models of DNA replication, over multiple generations:

Figure 5: Experimental predictions of three competing models of DNA replication over three generations.

And now, here are the actual observations of Meselson and Stahl (Figure 6):

Figure 6: Density gradient centrifugation of E. coli DNA over multiple generations. E. coli grown in 15 N DNA were switched to 14 N and then harvested at nine different time points. The DNA was centrifuged resulting in the banding pattern shown here.

As they let the bacterial cells grow and divide further, Meselson and Stahl observed that the 15 N DNA band disappeared, a band of 14 N DNA appeared and then got progressively darker, and a band of intermediate density appeared and persisted at about the same intensity. This strongly discredited the dispersive model of DNA replication , which predicted that only one band of DNA would exist and would get progressively less dense as the amount of 14 N DNA in the dispersive mixture increased with each generation . Thus, in one simple experiment with very clear results, Meselson and Stahl solidly disproved two of the possible models of DNA replication, while strongly supporting another.

• "The most beautiful experiment in biology"

The scientific community agreed that this was powerful evidence in support of the semi-conservative model . John Cairns, one of the leading molecular biologists of the era, called it, "the most beautiful experiment in biology." To this day, the Meselson and Stahl experiment is taught around the world as a classic example of the modern scientific method of experimentation. With one simple design, three scientific hypotheses were tested by the observation/verification of their predictions.

Scientists now have a detailed understanding of the molecular events of DNA replication (see our DNA III module). These molecular events occur just as predicted by the Watson and Crick model of DNA structure, and verify that the semi-conservative model of DNA replication is indeed correct. The Meselson and Stahl technique of labeling DNA strands with nitrogen isotopes is still employed by scientists around the world as they continue to explore the mysteries and complexities of DNA, the genetic material of life.

Table of Contents

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	two old strands. two new strands. one old and one new strand. two strands with variable proportions of new and old DNA. a variable number of old and new strands. N N (heavy) DNA, and after TWO generations in the N medium, cells will contain 25% N N DNA, 50% N N DNA, and 25% N N DNA. 50% N N DNA and 50% N N DNA. 50% N N DNA and 50% N N DNA. 50% N N DNA and 50% N N DNA. 25% N N DNA and 75% N N DNA. N N (heavy) DNA, and after THREE generations in the N medium, cells will produce the following band(s) in density-gradient centrifugation: a single band of DNA with medium density. one band of light DNA and one band of DNA with medium density. one band of heavy DNA and one band of DNA with medium density. one band of heavy DNA and one band of light DNA. three bands corresponding to DNA of light, medium and heavy density. True False N N (heavy) DNA, and after ONE generation in the N medium, cells subjected to density-gradient centrifugation will produce a single band of DNA with medium density. True False Search for: '); document.write(''); } // --> ( ) .'); } else{ document.write('This form changes settings for this website only.'); } //--> Send mail as: '); } else { document.write(' '); } } else { document.write(' '); } // --> '); } else { document.write(' '); } } else { document.write(' '); } document.write(' TA email: '); } else { document.write(' '); } } else { document.write(' '); } // --> Other email: '); } else { document.write(' '); } } else { document.write(' '); } // --> "Floating" navigation? '); } else if (floatNav == 2) { document.write(' '); } else { document.write(' '); } // --> Drawer speed: '; theseOptions += (glideSpeed == 1) ? ' ' : ' ' ; theseOptions += (glideSpeed == 2) ? ' ' : ' ' ; theseOptions += (glideSpeed == 3) ? ' ' : ' ' ; theseOptions += (glideSpeed == 4) ? 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Coli DNA Structure Meselson and Stahl Experiment Structural Basis of DNA Replication Bidirectional DNA Replication How Nucleotides Are Added in DNA Replication DNA Replication (E. coli) DNA Replication Fork Telomerase Function Direct Repair Nucleotide Excision Repair DNA Replication Methyl-directed Mismatch Repair Proofreading Function of DNA Polymerase Video Quiz - DNAs Dark Lady Mutations and Cancer DNA Structure 1 About the Authors Table of Contents Sample Chapter Supplements AP* Correlation AP* Instructor's Manual CPS eInstruction PowerPoint Files Premium Content - Lecture Outlines Premium Content - Instructor's Manual Premium Content - 8th Edition Changes Premium Content - Answers to Inquiry Questions Premium Content - Answers to EOC Questions  >   >   >  Please read our and before you explore our Web site. To report a technical problem with this Web site, please contact the . share this! August 29, 2024 This article has been reviewed according to Science X's editorial process and policies . Editors have highlighted the following attributes while ensuring the content's credibility: fact-checked peer-reviewed publication trusted source Scientists unlock the secrets of how a key protein converts DNA into RNA by Ali Sundermier, SLAC National Accelerator Laboratory Researchers at the Department of Energy's SLAC National Accelerator Laboratory have uncovered new insights into the fundamental mechanisms of RNA polymerase II (Pol II), the protein responsible for transcribing DNA into RNA. Their study shows how the protein adds nucleotides to the growing RNA chain. The results, published in Proceedings of the National Academy of Sciences , have potential applications in drug development. Pol II is found in all forms of life, from viruses to humans. Its role in gene expression , the process by which genetic information is used to synthesize proteins, makes it one of the most important proteins in the cell. Understanding the precise mechanism by which RNA polymerase adds nucleotides to RNA has been a longstanding challenge in the scientific community. Previous studies have provided only partial, low-resolution glimpses into this process. One of the major challenges in studying Pol II has been the transient nature of the metals, particularly magnesium, within its active site . These metals play a crucial role in the chemical reactions that drive nucleotide addition, but their fleeting presence makes them difficult to observe. "The chemistry of the polymerase involves metals that are transient in the active site, making them hard to see," said collaborator Guillermo Calero, a researcher and professor at the University of Pittsburgh. "This has been a significant obstacle in fully understanding the nucleotide addition process." To overcome these challenges, the research team used a novel crystallization technique that involved a special salt known for promoting protein-protein interactions. That technique allowed the researchers to capture the polymerase in a previously unseen state. This breakthrough allowed them to observe the "trigger loop," a mobile part of Pol II that positions nucleotides in the active site, in unprecedented detail. The use of SLAC's Linac Coherent Light Source (LCLS) X-ray laser was another key component of the study. It allowed the researchers to collect data before significant radiation damage occurred to the sample, providing a clearer picture of the polymerase's structure and function. "For the first time, we were able to see the three magnesium ions in the active site," said collaborator and SLAC scientist Aina Cohen. "This was only possible because of the free-electron laser data, which enabled us to see the extremely radiation-sensitive third metal ion." Another interesting finding emerged from studying a mutated version of Pol II. This mutant RNA polymerase operates faster than the wild type but also produces more errors. "The mutation changes the structure of Pol II," said collaborator Craig Kaplan, a professor at the University of Pittsburgh. "Using LCLS, we can identify these structural changes, which could reveal how the mutation impacts Pol II's activity." The team is already working on time-resolved experiments to capture the real-time dynamics of the polymerase's trigger loop as it interacts with nucleotides with the hopes of unraveling the complexities of RNA polymerase function and contributing to the broader understanding of gene expression. Further, by understanding the detailed mechanisms of human Pol II, researchers can now explore the development of molecules that could inhibit viral and bacterial polymerases while reducing harmful interactions with human polymerases. This is particularly relevant in the field of drug discovery, where the goal is to design drugs that are effective against pathogens but safe for human cells. "These structures not only advance our understanding of how human RNA polymerase functions, but they also provide a foundation to design more selective antiviral medications with less adverse side effects," Cohen said. 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Use this form if you have come across a typo, inaccuracy or would like to send an edit request for the content on this page. For general inquiries, please use our contact form . For general feedback, use the public comments section below (please adhere to guidelines ). Please select the most appropriate category to facilitate processing of your request Thank you for taking time to provide your feedback to the editors. Your feedback is important to us. However, we do not guarantee individual replies due to the high volume of messages. E-mail the story Your email address is used only to let the recipient know who sent the email. Neither your address nor the recipient's address will be used for any other purpose. The information you enter will appear in your e-mail message and is not retained by Phys.org in any form. Newsletter sign up Get weekly and/or daily updates delivered to your inbox. You can unsubscribe at any time and we'll never share your details to third parties. More information Privacy policy Donate and enjoy an ad-free experience We keep our content available to everyone. Consider supporting Science X's mission by getting a premium account. E-mail newsletter Open access Published: 29 August 2024 DNA hydrogels and their derivatives in biomedical engineering applications Wenting Li 2 , Pu Yang 1 , Naisi Shen 1 , Anqi Yang 1 , Xiangjun Liu 1 , Yikun Ju 1 , Lanjie Lei 3 & Bairong Fang 1   Journal of Nanobiotechnology volume  22 , Article number:  518 ( 2024 ) Cite this article Metrics details Deoxyribonucleotide (DNA) is uniquely programmable and biocompatible, and exhibits unique appeal as a biomaterial as it can be precisely designed and programmed to construct arbitrary shapes. DNA hydrogels are polymer networks comprising cross-linked DNA strands. As DNA hydrogels present programmability, biocompatibility, and stimulus responsiveness, they are extensively explored in the field of biomedicine. In this study, we provide an overview of recent advancements in DNA hydrogel technology. We outline the different design philosophies and methods of DNA hydrogel preparation, discuss its special physicochemical characteristics, and highlight the various uses of DNA hydrogels in biomedical domains, such as drug delivery, biosensing, tissue engineering, and cell culture. Finally, we discuss the current difficulties facing DNA hydrogels and their potential future development. Graphical Abstract Introduction Deoxyribonucleotide (DNA) is a natural biopolymer with controllable chemical structure and biological function, presenting substantial potential for development as a functional material [ 1 ]. As a natural biopolymer, DNA serves as the primary vehicle for encoding, storing, and transferring genetic information [ 2 ]. The DNA molecule consists of two single strands of deoxyribonucleotides that adhere to the Watson–Crick base-pairing principle. These DNA strands are organized in a particular coding sequence to create a stable double-helix structure [ 3 ]. This highly selective base recognition and sequence coding design capability endows DNA with excellent assembly capabilities. In 1982, Seeman designed the first linear DNA double helix. Since then, artificial DNA synthesis and modification techniques have been developed and matured, and DNA can be easily designed and synthesized from known sequences. DNA molecule applications have gradually expanded from their biological roles to the field of material science [ 4 ]. DNA molecules can be precisely programmed with simple chemical modifications to build materials with desired mechanical, biological, and structural properties [ 5 , 6 , 7 ]. For instance, the DNA manipulation and modification using specific instrumental enzymes has led to the development of multifunctional DNA nanostructural units [ 8 ]. Additionally, introducing a variety of functional groups at different positions in the DNA strand, such as sulfhydryl and amino groups [ 9 ], topologies can be changed in vitro using simple enzymatic reactions. Various compounds have been developed based on specific DNA structural and functional changes that exhibit potential biological uses in different areas of biomedicine [ 10 , 11 , 12 ]. Hydrogels are attractive biomaterials owing to their high biocompatibility and physical characteristics that resemble biological tissues. New hydrogels with unique functions are frequently reported [ 13 , 14 , 15 ]. Among the various polymer materials, researchers have been interested in using DNA to construct hydrogels. This is because DNA hydrogels not only retain the hydrogel backbone, but their unique programmability allows precise control of polymer chain interactions, thus, researchers are able to determine the formation and behavior of hydrogels in unique ways not possible with traditional hydrogel materials. When designing synthetic hydrogel structures, researchers can insert functional DNA nanostructures into the polymer network to control the behavior of the hydrogel in different environments. Examples include DNA aptamers with specific affinities for targets, i-motif structures that form at specific pH values, DNA enzymes that catalyze different chemical reactions, and more. These functional DNA structures provide hydrogels with sensitive stimulus responsiveness and offer more possibilities for the development of hydrogels. Additionally, the three dimensional (3D) scaffold of DNA hydrogels has a certain degree of mechanical rigidity that provides a large number of attachment sites, enhancing their function of stabilizing the immobilized substrate [ 16 ], while the introduction of other nanomaterials, such as magnetic and metal nanoparticles (NPs), into DNA hydrogels can improve their functional properties [ 17 , 18 ]. Moreover, compared with other synthetic polymer hydrogels, DNA is inherently biocompatible and is easily recognized by the human body, thus reducing practical application-related risks. Furthermore, the biodegradability of DNA eliminates the major concerns associated with most synthetic polymers [ 19 ]. DNA hydrogels offer many other advantages, such as specific molecular recognition, controlled phase transitions, and mechanical properties [ 20 , 21 , 22 , 23 ]. Different strategies have been devised to synthesize DNA hydrogels for biomedical engineering applications in various fields, such as biosensing, drug loading, tissue engineering, and cell culture. In this study, we provide an overview of significant recent advancements in DNA hydrogel technology. We outline the different design philosophies and methods of DNA hydrogel preparation, discuss their special physicochemical characteristics, and highlight the various uses of DNA hydrogels in biomedical domains. Finally, we elaborate on the difficulties facing DNA hydrogels and their potential for future development. Materials for DNA hydrogel synthesis Constructing abundant DNA strands is necessary for preparing DNA hydrogels. Whether relying on artificial synthesis or genome extraction, the cost of these two methods is high, and they often synthesize DNA strands of insufficient length and purity. To this end, researchers have developed various nucleic acid amplification methods to produce long DNA strands to meet research needs. Polymerase chain reaction (PCR), rolling circle amplification (RCA), and hybridization chain reaction (HCR) have been used to produce DNA hydrogels [ 24 , 25 ]. RCA and HCR are amplification methods at a constant temperature, simpler to perform and more efficient than PCR. RCA and HCR have different advantages. RCA is a simple and effective enzymatic amplification technique that is performed at constant temperatures and uses a minor quantity of circular DNA template [ 26 ]. This amplification method results in the continuous extension of DNA to form ultra-long linear DNA strands [ 27 , 28 , 29 ]. The hybridization chain reaction eliminates the complex thermal cycling step and achieves ultra-high detection sensitivity similar to PCR. As RCA does not require coenzymes, it is cheaper to synthesize DNA hydrogels. Further, non-linear HCR forms multiple branching DNA nanostructures. Triggered by promoter sequences, specific hairpin structures are opened to form self-assembled DNA nanostructures. Compared with other nucleic acid amplification methods, HCR has higher sensitivity and selectivity [ 30 , 31 ]. Functional DNA structures are important components of DNA hydrogels, such as i-motif structures, DNA enzymes, and aptamers, which provide molecular recognition to hydrogel polymers, further enriching the stimulus responsiveness of DNA hydrogels [ 32 , 33 ]. DNA aptamers are commonly used functional DNA structures, obtained by in vitro screening, the systematic evolution of ligands by exponential enrichment (SELEX), and random DNA sequences and can bind to various small molecules. Therefore, aptamers are mostly used as targeting ligands to assist DNA hydrogels in delivering therapeutic drugs, such as small RNA (siRNA), specifically to diseased cells, so that normal cells will not be damaged and the effect of targeting therapy can be achieved [ 34 ]. Nucleic acid aptamers present notable advantages, such as small physical size for easy transportation, customizable structure, high thermal stability, and ease of chemical modification. These qualities allow DNA aptamers to be used for exosome isolation and bioanalysis [ 35 , 36 ]. As biologically active molecules, DNA aptamers leave the physicochemical properties of the hydrogel unchanged and enable the DNA hydrogel to have a highly specific targeting function for various biomolecules [ 37 ]. Some DNA aptamers also undergo conformational changes in response to potassium ions, triggering a change in hydrogel volume, and can therefore be used for sensitive detection of potassium ions. [ 38 ]. In addition, these functional DNAs can act as cross-linking agents that allow the hydrogels to self-assemble at near-body temperature, making them ideal for encapsulating cells. DNA hydrogel construction There are many different ways to categorize DNA hydrogels. Generally, they are divided into two categories according to their composition: pure DNA hydrogels prepared entirely by DNA assembly, which are assembled by constructing various DNA modules and patterns, or by nucleic acid amplification to obtain long DNA strands entangled to form DNA hydrogels; hybrid DNA hydrogels, which usually contain other natural or synthetic polymers where the DNA acts as a cross-linking agent to cross-link and form hydrogels. In this section, we will discuss the synthesis strategies based on these two components of hydrogels and how they can be applied in different fields. Hybridized DNA hydrogels using DNA cross-linking Hybridized DNA hydrogels are made by cross-linking DNA with other polymers. For hybridized DNA hydrogels, the hydrophilic polymer chains act as scaffolds for the main body of the gel, and the DNA chains mainly act as cross-links during the gelation process, which is significantly different from that of pure DNA hydrogels. The first reported DNA hybridization hydrogel was synthesized by Nagahara and Matsuda in 1996 as a polyacrylamide-DNA hybridization hydrogel [ 39 ]. They crosslinked short DNA sequences modified with acrylates to polyacrylamide polymer chains. The authors then showed that there are two ways to achieve gelation; by hybridizing the DNA sequences with two other DNA strands attached to the polymer backbone and by attaching complementary DNA branched strands directly to the polymer backbone without adding external DNA connectors. Following this work, polyacrylamide hydrogels with different DNA cross-links have been produced. For example, Willner et al. [ 40 ] designed pH-controllable shape memory hydrogels. These two nucleic acid chains act as “connectors” that aggregate into an i-motif structure at pH 5.0 to form a stable hydrogel, and then dissociate from the i-motif structure at pH 8.0 to turn the hydrogel into a liquid (Fig.  1 A). Also utilizing the i-motif as a cross-linking unit, Guo et al. [ 41 ] designed DNA hybridization hydrogels as thin-film structures; then, i-motif sequences were inserted into the active layer of bilayer polyacrylamide to direct the programmable stimulus response and reversible shape deformation of the hydrogels. As shown in Fig.  1 B, the formation of the i-motif structure was induced by pH adjustment, thus changing the cross-linking density of the active layer and becoming swollen, while the passive layer, which was tightly adhered to the active layer, remained unchanged in volume, which induced the bending deformation of the hydrogel film. Liao et al. [ 42 ] proposed another method for assembling stimulus-responsive DNA-polyacrylamide hydrogels to stabilize microcapsules. They used HCR to generate a DNA crosslinked hydrogel coat encapsulating CaCO 3 particles. Due to the incorporation of cofactor-dependent DNAzyme units, the stiffness of the outer hydrogel decreases and porosity increases when the crosslinked DNAzyme substrate is cleaved by the corresponding cofactor, thereby releasing the loadings. Constructing a hydrogel based on a similar strategy, Sun et al. [ 43 ] developed a novel stimulus-responsive aptasensor. The DNA-acrylamide hydrogel formed by two polyacrylamide chains was functionalized by DNA hairpins and involved in chain-induced HCR. Upon fumonisin B (FB) exposure, the complexes formed by FB with the DNA functional units promote the dissociation of the crosslinked bridging units, leading to the disintegration of the encapsulated metal-organic framework (MOF) hydrogel shell. These polyacrylamide-based hybrid DNA hydrogels use DNA units as “bridges” to form hydrogels that undergo a solution-gel transition as the DNA polymerizes and dissociates. The properties of the polymer itself, such as chemical flexibility and stability, are well preserved in these hybrid hydrogels. In addition, other organic backbone materials have been gradually explored, and various hybridized DNA hydrogels, such as proteins and peptides, have been prepared [ 44 ]. Li et al. [ 45 ] prepared supramolecular peptide-DNA hydrogels using “X”-shaped DNA as a cross-linking agent (Fig.  1 C). Under physiological conditions, the gelation process was rapid. Moreover, this hydrogel presents self-repairing ability. The peptide-DNA hydrogel blocks are modified into different colors for easy observation, they stick together upon contact, and after a few minutes, the fully bonded gel blocks can be picked up with tweezers. They can even be completely fused into a homogeneous whole. This is because DNA grafted polypeptides have the same cross-linked “sticky ends” as the “X-shaped” DNA linkers, so when they come into contact, the “sticky ends” of the “X-shaped” DNA linkers in one of the hydrogels dissociate from the polypeptide chain and form a new double strand with the DNA “sticky ends” on the polypeptide chain of the other hydrogel. Inorganic nanomaterials, such as magnetic and carbon nanotube metal NPs [ 46 , 47 ], have also been used to create hybridized DNA hydrogels in order to enhance their performance and give them additional functionality. For example, magnetic nanoparticles (MNPs) can form dynamic cross-linking points in the hydrogel network through physical entanglement. Yang et al. [ 48 ] developed a magnetically driven soft robot based on DNA hydrogel by incorporating MNPs into the hydrogel. RCA first amplifies the long ssDNA, and short ssDNA-modified MNPs are attached to the long ssDNA as cross-linking sites. These cross-linking sites change position as the long ssDNA strand slides, and short ssDNA also serves as a primer for the long-stranded ssDNA, triggering re-amplification. The long-stranded DNA is entangled to form a stable magnetic hydrogel network, and the navigational motion of the DNA hydrogel is driven by MNPs. The magnetic nanomaterials have a synergistic effect with the DNA, making the hydrogel highly adaptable. For example, it is shape-adaptive, allowing it to change shape flexibly in various environments, and can be used to efficiently transport molecular drugs. DNA gelation with a variety of materials to form hydrogels is mainly achieved using chemical cross-linking and physical entanglement, as opposed to chemical cross-linking, which has a permanent and irreversible covalent effect. Physical cross-linking is relatively dynamic and flexible [ 49 , 50 ]. However, Tang et al. [ 51 ] showed that DNA hydrogelation is possible by DNA strands and upconversion nano-particles (UCNPs) to prepare hybridized DNA hydrogels. The distance between DNA strands (negatively charged) and UCNPs (positively charged) is shortened by electrostatic attraction. Through electrostatic interactions (EIs), UCNPs and DNA strands bind together at the interface to form a hydrogel network. Remarkably, this process results in the formation of a large number of hydrogels in only 1 s, providing a new paradigm for the preparation of DNA hydrogels. In summary, hybridized DNA reduces the concentration of DNA required for gelling, which alleviates the disadvantages of rate-pure DNA-based hydrogels such as limited stability, high synthesis cost, and low yield, and scales up production. However, synthetic polymer materials within the hybridized DNA hydrogels can still limit hydrogel biocompatibility. Therefore, this should be considered when selecting suitable polymer materials for cross-linking to form hydrogels. ( A ) pH-stimulated DNA hydrogels with shape memory properties. Adapted reprinted with permission from Ref [ 40 ]. Copyright 2014, Wiley. ( B ) Smart bilayer polyacrylamide/DNA hybrid hydrogel. Adapted reprinted with permission from Ref [ 41 ]. Copyright 2020, Wiley. ( C ) Peptide-DNA hydrogel with multiple modification sites. Adapted reprinted with permission from Ref [ 45 ] Copyright 2014, Wiley Pure DNA hydrogels prepared based on DNA self-assembly enzymatic cross-linking Pure DNA hydrogels are synthesized using DNA as the only component of the hydrogel, and there are two strategies for synthesizing them: one is to use DNA strands as building blocks, which are self-assembled or enzymatically ligated and cross-linked to form a polymeric network. The other is to directly wind long DNA strands from nucleic acid amplification into a hydrogel. Self-assembling DNA hydrogels require multiple building blocks with repetitive sequences, usually linear and branched DNA structures. In general, branched DNA structures are more customizable and ordered than linear DNA, but whether linear or branched, these building blocks usually contain special sticky end structures with complementary nucleotide sequences that form hydrogen bonds. Therefore, the networks of these DNA hydrogels are formed by recognizing and assembling the sticky ends of DNA. In 2006, Luo et al. reported hydrogels made entirely of branched DNA, which was the first time DNA hydrogels were formed by complementary hybridization of sticky ends. Rational self-assembly of ssDNA strands produces various branched DNA modules, such as X-type or T-type DNA [ 52 ]. As shown in Fig.  2 A, various branched DNA modules, i.e., X-type, T-type, or Y-type DNA, were generated by the rational self-assembly of ssDNA strands. Each branched DNA strand had a different number of complementary sticky ends, which hybridized and combined with each other, guiding the branched DNA into a network and forming a stable DNA hydrogel. For the first time, the entire preparation process was accomplished under physiological conditions. The advantage of this method is that it is simple and inexpensive, but a large number of palindromic sequences are used in the preparation process, resulting in inhomogeneous DNA hydrogels. Therefore, Liu et al. [ 53 ] assembled DNA hydrogels using a Y-type scaffold and a linker (Fig.  2 B). The Y-type scaffold consists of three ssDNA strands and a linker, which is a linear double strand composed of two ssDNA strands. The Y-scaffold and linker are also designed with sticky ends for complementary cross-linking, but their sticky ends minimize the use of palindromic sequences so that the DNA hydrogel formed can be more homogeneous. Additionally, enzymes are important molecular tools that aid in sticky end joining. The main enzymes used to construct DNA hydrogels are DNA ligases and polymerases. Ligases repair gaps in the assembly of branched DNA. Luo et al. proposed another novel cellular protein synthesis method to synthesize DNA hydrogels [ 54 ]. They added plasmid DNA to the hydrogel and then used T4 DNA ligase to join the gaps between the X-DNA and the linear plasmid to form a DNA hydrogel network. This DNA hydrogel is mainly used for efficient cell-free production. Notably, unlike other polymerases that require templates and primers to synthesize DNA strands, terminal deoxynucleotidyl transferase (TdT) catalyzes the synthesis of DNA from random mononucleotide dNTP only in the presence of primers, which greatly improves the efficiency of DNA hydrogel preparation [ 55 , 56 ]. Hydrogels with 3D network structures can also be prepared by using nucleic acid amplification to obtain long DNA strands. Wang et al. [ 57 ] used clamp HCR to prepare self-assembled DNA hydrogels (Fig.  2 C). Three DNA strands are involved in this system, and unlike other long DNA strands that spontaneously form homogeneous hydrogels, the DNA initiator induces a sol-gel transition that controls the hydrogel in three-dimensional space and time. This hybridization reaction, precisely triggered by the initiator strands, offers the possibility of constructing custom-shaped DNA hydrogels. RCA is another stable and rapid method of nucleic acid amplification that can be used to obtain long DNA strands. In the process of successive replication of DNA sequences to form long strands of DNA, the physicochemical properties of the polymerization network can also be improved by the addition of different repetitive functional sequences and complementary regions, thus enabling the functionalization of the hydrogel. Guo et al. [ 58 ] synthesized DNA network structures with different crystallinity based on RCA (Fig.  2 D) and controlled the crystallinity of inorganic magnesium pyrophosphate (MgPPi) by adjusting the conditions of RCA to change the size of the hydrogel network gaps. Both RCA and HCR are thermostatic amplification methods that are simple and efficient. Using these amplification methods, repetitive functional DNA sequences can be obtained, and as with hybridized DNA hydrogels, insertion of these functional DNA structures into a polymer network allows for the preparation of DNA hydrogels that are responsive to a variety of factors [ 59 , 60 , 61 ]. Hu et al. [ 62 ] synthesized smart DNA hydrogels using single-stranded DNA capable of assembling functional units. Under slightly acidic conditions, the ssDNA strands self-assemble to form a linear DNA structure containing an i-motif structure, which then naturally breaks down under slightly alkaline conditions, leading to cross-linking of the hydrogel. Therefore, the DNA hydrogel can be reversibly transformed from hydrogel to solution in pH-controlled conditions. In summary, the self-assembly strategy of DNA modules based on the sticky end makes the formation and post-formation behavior of hydrogels more controllable, and the efficient nucleic acid amplification can in turn provide us with a large amount of nucleic acids while reducing the cost. Moreover, compared to hybridized DNA hydrogels, pure DNA hydrogels without the use of other polymers are not only chemically similar to DNA molecules with good biocompatibility and enzyme degradation, but also show good biodegradability, precise structural controllability, and responsiveness to specific stimuli, which is promising for biomedical fields. ( A ) DNA hydrogels assembled from DNA building blocks. Adapted reprinted with permission from Ref [ 52 ]. Copyright 2006, Springer Nature Ltd. ( B )Preparation of DNA hydrogels by of Y-scaffolds and linkers. Adapted reprinted with permission from Ref [ 53 ]. Copyright 2010, Wiley. ( C ) DNA initiator-induced HCR and gelation process. Adapted reprinted with permission from Ref [ 57 ]. Copyright 2017, Wiley. ( D ) RCA-based DNA hydrogels. Adapted reprinted with permission from Ref [ 58 ]. Copyright 2023, Elsevier Ltd Physicochemical properties of DNA hydrogels Among many hydrogel materials, DNA hydrogels have outstanding mechanical properties and stimulus responsiveness. Different textures of DNA hydrogels perform unique functions. For example some DNA hydrogels can be used as intelligent soft robots [ 63 ], and others with higher mechanical strength are used as cartilage substitutes for cartilage repair [ 64 ]. The mechanical strength of DNA hydrogels is related to the construction method and hydrogel composition. In general, DNA hydrogels assembled from DNA modules have a higher shear modulus than hydrogels formed from continuously elongated ultralong DNA strands, indicating that the latter are mechanically stiffer [ 65 ] and the former can exhibit higher elasticity, such as ssDNA hydrogels constructed on the basis of the RCA reaction [ 28 , 66 ]. This is due to the physical entanglement between the ultra-long ssDNA, and DNA hydrogels are usually soft in texture. Such DNA hydrogels have good shape adaptation, making up injectable DNA hydrogels with good thixotropic properties [ 64 ]. Physically crosslinked DNA hydrogels are mainly stabilized by various non-covalent interactions. In contrast to the high strength and stable covalent bonds, these non-covalent bonds reversibly break and form by the external environment [ 67 , 68 ]. Wang et al. [ 69 ] prepared pure DNA hydrogels with different concentrations of hydrogen bonds (Fig.  3 A). They prepared three sets of DNA hydrogels based on double RCA and self-assembly, in which the ultra-long single-stranded DNA precursors contained different amounts of hydrogen bonds. The experimental results showed that the higher degree of hydrogen bonding in the precursor DNA, the denser the network inside the hydrogel, the higher the mechanical properties, and the better the capture efficiency. On the other hand, DNA hydrogels based on chemical cross-linking are usually more stable, and the range of hydrogel applications, such as shear-thinning and injectable properties, can be further expanded by introducing dynamic covalent bonds. This is due to the fact that dynamic covalent bonds open under shear and can be spontaneously reconnected after the shear force is removed [ 70 ]. In addition, rational design of the backbone structure of DNA hydrogels is another effective approach. Liu et al. [ 71 ] assembled a DNA double cross (DX) backbone rigid hydrogel. The rigidity of the DNA double helix can improve the polymerization in the kinetic interlocking multiple unit (KIMU) strategy, so they designed the DNA DX single strand as the DX backbone (Fig.  3 B), on which the DX supramolecular polymer was prepared with high molecular weight and high stability. Finally, the supramolecular hydrogels further constructed by utilizing DX polymers as rigid backbones have ultra-high mechanical strength. Similarly, Yang et al. [ 72 ] developed a new L-DNA hydrogel (Fig.  3 C). The L-DNA hydrogel exhibits superior biostability in comparison to the mirror-image isomer deoxyribose, and after 30 days of co-cultivation with fetal bovine serum, there is no discernible loss in mechanical strength. Moreover, it does not cause the body to manifest an inflammatory response. In 2024, Shi et al. [ 73 ] synthesized three DNA scaffolds with different shapes and sequentially increasing stiffness, connected them with DNA linkers of different stiffnesses, and formed hydrogels with simple mixing. They further revealed the close relationship between the rigidity and structure of DNA hydrogels. Furthermore, during the synthesis of hydrogels, by adjusting the ratio of hairpin chains in the hybridization chain reaction, the mesh size of the hydrogel can be altered to meet various clinical needs [ 74 ]. ( A ) Schematic representation of DNA hydrogels prepared from long-stranded DNA with different degrees of hydrogen bonding. Adapted reprinted with permission from Ref [ 69 ]. Copyright 2023 Biosensors ( B ) Schematic representation of DNA double cross-linking hydrogels. Adapted reprinted with permission from Ref [ 71 ]. Copyright 2022, Wiley. ( C ) D-DNA and L-DNA hydrogels with special strengths. Adapted reprinted with permission from Ref [ 72 ]. Copyright 2021, Wiley Stimulus responsiveness of DNA hydrogels refers to the behavior of base complementary pairing between DNA strands or functional nucleic acid structure changes triggered by various factors, which affects the volume of the hydrogel or changes in physicochemical properties [ 75 ]. Sequences of functional DNA units can be introduced to programmatically alter the response properties of hydrogels to exhibit dynamic volume changes in response to targets. Among them, hydrogels with pH-responsive behavior usually contain special structures such as i-motif structure, T-A-T triple helix structure, and C-G-C + triple helix structure [ 76 , 77 , 78 ]. Among them, the i-motif is a cytosine C-rich structure, and after being protonated, it becomes sensitive to pH changes. Therefore, special DNA functional modules can be used to trigger the solution-gel transition in hydrogels. Liao et al. [ 60 ] designed stimulus-responsive DNA hydrogel microcapsules, where the hydrogel consisted of a functional DNA structure, an i-motif structure, and a polyacrylamide, and under acidic conditions, the i-motif structure formed a “connecting bridge” that separated the i-motif-connected double-stranded units, which led to the separation of the microcapsule shell. Nucleic acid aptamers are another commonly used functional unit of DNA that specifically recognizes and binds to target molecules. When the aptamer binds to the target molecule, the structure of the hydrogel network is changed [ 79 ]. DNA enzymes do the same by disintegrating the substrate strand to disintegrate the DNA network structure, dissolving the hydrogel. Gao et al. [ 80 ] also methylated the edges of the DNA tetrahedra with DNA adenine methyltransferase (Dam), and methylation-sensitive restriction endonucleases then cleaved the DNA tetrahedra to release amyloglucosidase, which catalyzes glucose production, and finally quantitative readings were taken using PGM. Li and Yang [ 81 ] developed a handheld glucometer based on a multicomponent nuclease-based DNA hydrogel. When the target miRNA is added, the active nuclease triggers the hydrogel to break down and release the encapsulated amylase. Metal ion-dependent DNAzyme binds to metal cofactors and activates DNAzyme to degrade the substrate. Various DNAzyme-based DNA hydrogels have been reported for metal ion detection. Guo et al. [ 82 ] developed a bilayer DNA hydrogel membrane. The lead (Pb 2+ ) or uranyl (UO 22+ ) ions can activate the DNA enzyme to cleave the substrate strand and release the negatively charged cleavage fragments. The negative charge density of the active layer decreases and shrinkage occurs, which triggers the large macroscopic shape of the bilayer hydrogel membrane to change significantly. This DNA hydrogel combines target introduction, signal amplification, and signal output to build a smart offloading system of biosensors for rapid detection of target molecules or drugs to meet the requirements of clinical portability and sensitivity [ 83 , 84 , 85 ]. There is a tendency to develop dynamic hydrogels in which various factors modulate the change in hydrogel structure. For example, Quan et al. [ 86 ] added cations to the hydrogel to control cross-linking and disassembly between DNA strands and spermine. The most straightforward approach is to introduce a response structure. Such structures can be responsive DNA structures or responsive polymer chains [ 87 , 88 ]. Designing these different functional DNA hydrogels as smart sensors has attractive applications in the field of bioengineering. Biomedical application of DNA-based hydrogel construction Drug delivery and therapy. Controlled release and targeted delivery of therapeutic drugs are important issues in modern biomaterial research. Conventional drug delivery systems usually suffer from low drug bioactivity and unsatisfactory therapeutic effects owing to systemic toxicity, repeated administration, and changes in the internal environment. Moreover, the difficulty in precisely controlling drug targeting and release often results in insufficient efficacy or severe side effects. Therefore, finding an ideal mode of drug delivery is critical. DNA hydrogels can be used as a carrier for topical drug delivery, delivering high doses of active biomolecules to the target site continuously and slowly [ 89 , 90 , 91 ]. A suitable scaffold that can prevent the drug from spreading beyond the treatment site during administration to avoid side effects while simultaneously ensuring that the drug is released slowly at a certain rate to maintain its therapeutic activity is urgently required. The porous microstructure and cross-linking network of the DNA hydrogel can effectively bind to the drug molecule. In addition, by integrating stimulus-responsive structures into the DNA backbone or DNA junctions of the DNA hydrogel, the DNA hydrogel disintegrates under specific triggers, thus ensuring that the drug can be released exactly at the target site [ 92 , 93 ]. Li et al. [ 94 ] developed a multifunctional hydrogel (Agevgel) based on DNA scaffolds, in which the DNA strands act both as shape-variable scaffolds for loading immunomodulatory M2 macrophage-derived extracellular vesicles (M2EVs) and as antimicrobial building blocks (Fig.  4 A). The adherent DNA hydrogels not only ensure the time-dependent sustained release of silver nanoclusters (AgNCs) and M2EVs, but also serve as artificial extracellular matrices suitable for different shapes of diabetic alveolar bone defects (DABDs) and avoid unfavorable external environmental factors. Another strategy is to use the enzymatic action of the DNA hydrogel to disintegrate the hydrogel structure, resulting in a slow release of the encapsulated drug. Zhang et al. [ 95 ] used an enzyme-responsive DNA hydrogel (DSH) as a metformin (MET) delivery vehicle for the treatment of osteoarthritis (OA). With the degradation of DSH by DNAzyme, MET was slowly released into the joint cavity. This approach protected MET from rapid clearance by synovial fluid, and exerted a greater anti-inflammatory effect. In addition to delivering various types of small molecule drugs, the injectable DNA hydrogel can be designed to contain immunostimulatory motifs that bind to pathogen pattern recognition receptors, inducing an immune response. This promotes immune activation of the vaccine in vivo to enhance vaccine efficacy. Guo et al. [ 96 ] developed a nanotoxin-embedded DNA hydrogel. The DNA hydrogel contains both immunostimulatory CpG sequences and is enriched with guanine that can form a G-quadruplex structure, which stabilizes the structure of the DNA hydrogel and prolongs the retention time of the nanotoxin. Traditional cancer immunotherapy often suffers from low immune response rates and poor targeting. DNA hydrogels have excellent targeting capabilities, and they can be loaded with immunotherapeutic agents, chemotherapeutic agents, phototherapeutic agents, and other agents to the tumor site, thus allowing for the precise controlled release of drugs and triggering long-term anti-tumor effects [ 97 ]. However, these tumor immunotherapy monotherapies may suffer from insufficient immune activation and unsatisfactory immunosuppressive effects. DNA hydrogel-mediated combinatorial immunotherapies can play an important role in enhancing therapeutic efficiency. In 2024, Yang et al. [ 98 ] developed a smart DNA hydrogel (Fig.  4 B). The hydrogel is constructed from two ultra-long DNA strands containing three complementary functional units. One DNA strand is designed to contain an aptamer and an immunostimulatory sequence, CpG, which is used to load exosomes with antitumor effects, and the other DNA strand contains a multivalent G-quadruplex, which is used to load photodynamic agents. Additionally, in order to exert the combination immunotherapeutic effect, restriction endonuclease sites are designed between the functional units, and the hydrogel is stimulated to break down and release the functional units from the tumor location. The outcomes of the experiment demonstrated that the DNA hydrogel effectively activated the immune system, killed tumors, and dramatically prevented tumor growth. ( A ) Multifunctional hydrogel for delivery of extracellular vesicular DNA promotes reconstruction of diabetic alveolar bone defects. Microscopic CT images of the treatment group in the area of the bone defect after treatment. Adapted reprinted with permission from Ref [ 94 ]. Copyright 2023, Wiley. ( B ) DNA hydrogel-mediated combination immunotherapy loaded with natural killer cells and photodynamic agents. Adapted reprinted with permission from Ref [ 98 ]. Copyright 2024, Wiley Aptamers present a good choice for targeted transportation. A DNA-containing hydrogel can accurately bind to the target and target cancer cells, making chemotherapeutic molecules more drug-toxic and tumor-specific, thus increasing efficacy while minimizing multidrug resistance and side effects. Lee et al. [ 99 ] developed an immune checkpoint blocking DNA aptamer hydrogel (PAH). The DNA strands were designed to contain DNA of the programmed death receptor PD-1 aptamer and a single-stranded guide RNA sgRNA targeting sequence. In this way, CRISPR-associated protein 9 (Cas9) is able to exactly recognize and cut the sgRNA-guided DNA double-stranded structure. When Cas9 binds to the sgRNA, it triggers the hydrogel to degrade and release the PD-1 DNA aptamer, resulting in an effective anti-tumor effect over a long time. However, the retention time of this hydrogel was only a few days at the injection site; thus, multiple treatment repetitions were required, and the immunogenicity problem has not yet been solved. In 2023, Zhu et al. [ 100 ] used direct self-assembly of DNA molecules to fabricate DNA nanogels (DNGs). The preparation method is simple, and the DNGs are highly stable against physical forces and can be stored in concentrated solutions or powders for long periods of time. As shown in Fig.  5 A, by encoding specific DNA aptamers onto dendritic DNA molecular branches and encapsulating the chemotherapeutic drug doxorubicin, DNG can selectively target cancer cells to enhance chemotherapeutic drug efficacy and tumor specificity. Thus, the therapeutic efficacy can be improved while minimizing the side effects. Additionally, DNA hydrogels are prepared as smart carriers that respond to various external and internal stimuli [ 101 ], which not only improves drug efficacy but also minimizes cytotoxicity. In recent years, photothermal therapy has been gradually applied to the local treatment of tumors. The hydrogel network structure can be loaded with photothermal nano-agents and chemotherapeutic agents, and the development of DNA hydrogels with photothermal properties can realize efficient controlled drug release [ 102 , 103 ]. Guo et al. [ 104 ] established a photothermal-chemotherapeutic synergistic cancer treatment system by combining DNA hydrogels with MXene nanosheets. As shown in Fig.  5 B, hybrid DNA hydrogel with temperature-induced solution-gel transition was first prepared, and then the photothermal MXene nanosheets would be uniformly dispersed within the hydrogel. Under near-infrared light (NIR) irradiation, the temperature of the nanosheets increased, which induced the disintegration of the DNA double-stranded cross-linking structure, thus triggering the transformation of the hydrogel matrix into a solution. The DNA double strands were re-cross-linked and reformed into a hydrogel matrix after the NIR irradiation stopped. This property can also be used to design different shapes of hydrogels using models. Experimental results showed that DNA hydrogels loaded with the therapeutic agent doxorubicin were effectively released in a murine tumor model, causing direct damage to the tumor tissue, thus demonstrating efficient therapeutic properties against local cancer. ( A ) Targeted DNA hydrogel for slow release of doxorubicin. Adapted reprinted with permission from Ref [ 100 ]. Copyright 2024, Elsevier Ltd. ( B ) MXene-DNA hydrogel for light-triggered localized photothermal chemotherapy for the treatment of rhabdomyosarcoma mice, where the hydrogel undergoes adaptive shape changes according to the shape of the mold in the presence of near-infrared radiation. Adapted reprinted with permission from Ref [ 104 ]. Copyright 2022, Wiley When a stimulus-responsive DNA molecule dissociates or undergoes a conformational change upon binding to an external analyte, the structure of the hydrogel changes accordingly, releasing the probe encapsulated in the gel [ 105 , 106 , 107 , 108 ]. The detection process converts the target input into various physical or chemical outputs (mechanical, acoustic, optical, and electrical signals), thereby transforming various analytes into easily processed sensing signals for biosensing. DNA hydrogel sensors were developed based on their ability to respond to a variety of stimuli. Additionally, DNA hydrogels are good platforms for encapsulating catalytic substances, which are released to further catalyze the reaction [ 109 ]. The release of catalytic substances can further catalyze the reaction to produce amplified output signals [ 110 , 111 , 112 ]. The most common functional unit of stimulus-responsive DNA hydrogels is the aptamer, and the synthesis of sensitive biosensors using a variety of aptamers that bind to the molecule to be tested is a conventional strategy for the preparation of DNA hydrogels applied to biosensing [ 113 ]. Researchers have also developed a fluorescent DNA hydrogel system for prostate-specific antigen (PSA) detection [ 114 ]. Y-type DNA is used as a building block and is designed to be enriched with C-sequences to serve as a substrate for AgNCs with optical properties, whose fluorescence emission increases due to aggregation-induced emission (AIE) and a hydrogel structure that facilitates the formation of highly fluorescent signals [ 115 ]. These act as cross-linking agents to form dense hydrogels, which insulate AgNCs from environmental influences and produce strong fluorescence emission. When the target PSA binds to its specific aptamer, the DNA network structure disintegrates and the hydrogel collapses and dissolves, thus reducing the emission intensity. Also inspired by the excellent properties of DNA aptamer hydrogels, several target-responsive hydrogels have been designed as monitoring devices for ochratoxin A (OTA) detection. The existing single-mode OTA monitoring strategies are susceptible to various factors such as the environment, instrumentation, and operation, and the reliability and accuracy of the detection results requires improvement [ 116 ]. Therefore, Fan et al. [ 61 ] created a heme-based CuNCs-modified DNA hydrogel sensor for ochratoxin. Colorimetric detection is both sensitive and quick. OTA aptamers were used to create DNA hydrogels, which were then embedded with heme, cross-linked, and in situ encapsulated with fluorescent copper nanoclusters (CuNCs). When OTA appeared, the DNA aptamer formed a G-quadruplex structure by preferential binding. This enabled fluorescence and caused the CuNCs to burst. After OTA competitively attaches to the aptamer to create a G-quadruplex structure, the network structure of the DNA hydrogel is dissociated, causing hemoglobin to be released and CuNCs to fluoresce. The signaling cascade is amplified when the liberated heme attaches to the G-quadruplex to generate a DNAzyme that enables the CuNCs to fluoresce again. However, these aptamer-based hydrogels require “one-to-one” binding to the target, which can lead to less sensitive detection. Nucleic acid amplification techniques such as HCR, RCA, etc., have also been used for various signal amplifications, but these amplification strategies increase the sensitivity while being limited by the high concentration of DNA as well as the stringent conditions such as temperature and pH. Some DNA polymers such as G-quadruplexes are not only able to bind to the target, but also amplify the signal by reassembling into a special structure under the action of triggers to connect with the biosensor substrate [ 117 ]. Lu et al. [ 118 ] developed another approach to enhance signal sensing. The DNA hydrogel consists of layers of micropores that are interconnected to help enhance signal transmission. Moreover, the hydrogel makes it easier to monitor the sensed signals by connecting it to a smartphone sensing platform. Metal ion-dependent DNA enzymes are reaction units and cross-linkers in hydrogels and can be used for a variety of metal ion assays. Jiang et al. [ 119 ] developed a smart DNA hydrogel capillary sensor to convert Pb 2+ concentration into macroscopically visible changes in solution behavior (Fig.  6 A). The Pb 2+ -dependent DNA enzyme is the response unit and cross-linking unit of the hydrogel. The capillary is fixed at the origin of the calibration scale, and the crosslinked and stabilized hydrogel membrane completely blocks the capillary to prevent solution inflow. In the presence of Pb 2+ , the crosslinker substrate strand is cleaved by the activated DNA enzyme, the pores of the hydrogel membrane at the end of the blocked capillary enlarge and break, and the solution flows into the tube. The concentration of Pb 2+ is quantified by reading the distance and duration of the solution in the capillary. In this way the DNA hydrogel membrane translates the Pb 2+ concentration into a visualization of the flow behavior of the solution in the tube to facilitate detection. The electrochemical sensing properties can also be used to detect small molecules, providing valuable information for the diagnosis of a variety of diseases. For example, Yao et al. [ 120 ] developed an electrochemical sensing strategy by combining the hybridization chain reaction-activated Cas12a enzyme with DNA hydrogels. As shown in Fig.  6 B, the target protein binds to a specific region of DNA to form a complex that protects the DNA from being digested by nucleic acid exonuclease, and simultaneously, the long double stranded DNA produced by HCR activates Cas12a enzyme, which cleaves the DNA junction of the crosslinked DNA gels and releases a large amount of electroactive substances embedded in the gel, which exhibit highly amplified current signals under specific conditions, thus achieving signal amplification and sensitively detecting nuclear factors. Guo et al. [ 121 ] proposed a new nano-impact electrochemical (NIE) sensing strategy to prepare highly sensitive DNA hydrogels based on CRISPR technology. AgNPs were encapsulated within the DNA hydrogel to avoid adhesion of NPs on the electrode surface. The disintegrated hydrogel releases AgNPs that collide with the electrode material, resulting in a significant electrochemical signal. The sensitivity of the DNA hydrogel assay is very high, and the detection limit of this NIE biosensing strategy for miR-141 is as low as 4.21 aM, which is highly specific for practical applications. The drawback of limiting the sensitivity because of the low effective collision frequency was improved. Electrochemiluminescence (ECL) is another emerging effective analytical method for the sensitive determination of biomolecules, which has garnered substantial attention. Zhao et al. [ 122 ] constructed a target-induced DNA hydrogel biosensing platform. miRNA let-7a triggered strand displacement amplification, and the product then underwent cyclic amplification and induced HCR to generate dendritic DNA hydrogel structures. A positively charged amphiphilic perylene derivative (PTC-DEDA) was then intercalated into the DNA grooves of the hydrogel. PTC-DEDA, as the core of the ECL reaction, has a very high binding stability with the DNA hydrogel, which enables the dendrimer to generate a stable ECL reaction and thus obtain a strong ECL signal. Proteins and nucleic acids are used as biomarkers to reflect the health changes of the organism, and sensitive detection of abnormal activities of DNA, miRNA, or nucleic acid-related enzymes provides important information for the development of various diseases [ 123 , 124 , 125 , 126 ]. Chen et al. [ 125 ] constructed a hybrid DNA hydrogel for the detection of creatine kinase (CK-MB) by combining the technical amplification technology (EXPAR) and the CRISPR/Cas14a system. As shown in Fig.  6 C, CK-MB dissociates the aptamer-DNA complex by competitive magnetic separation, and the DNA strand forced to dissociate from the aptamer initiates the EXPAR system to generate the target ssDNA, which in turn activates the cleaving enzyme activity of Cas14a to disintegrate the hydrogel network. Thus, metal-organic framework nanosheets coated with platinum nanoparticles (PtNPs) decorated on the hydrogel were released and detected. The detection limit of the system for CK-MB was 0.355 pM, which is far below the clinically abnormal detection value. To achieve rapid, easy, and portable instant detection, microfluidic chips and microneedle patches around DNA hydrogels are becoming a new research hotspot. Yang et al. [ 127 ] reported a DNA hydrogel microneedle (MN) array based on strand substitution to achieve limiting signal amplification for the rapid detection of interstitial skin fluids (ISF). As shown in Fig.  6 D, the microneedle patch can easily penetrate the skin to reach the dermis, and when the target miRNA is present in the skin mesenchyme, a substitution reaction occurs within the DNA hydrogel. The DNA strand modified by the quenching group is replaced by the miRNA to produce a fluorescent signal, and then the DNA strand with the fuel probe immediately replaces the miRNA again, which is released to continue to induce the next substitution. This cycle ensures that enough ISF can be extracted in a short time for miRNA detection. This invasive sampling method provides a new idea for ISF extraction. ( A ) Schematic of DNA hydrogel-based Pb 2+ capillary sensor. Adapted reprinted with permission from Ref [ 119 ]. Copyright 2020, Elsevier Ltd. ( B ) DNA hydrogel-integrated electrochemical sensing method for detection of NF-κB p50. Adapted reprinted with permission from Ref [ 120 ]. Copyright 2022, Elsevier Ltd. ( C ) Detection of CK-MB system based on Cas14a cleaved DNA hydrogel. Adapted reprinted with permission from Ref [ 125 ]. Copyright 2021, Wiley. ( D ) Construction scheme and characterization data of DNA hydrogel-encapsulated microneedle arrays for sensing miRNA cross-linked microneedle patches [ 127 ]. Copyright 2022, American Chemical Society Tissue engineering DNA hydrogels not only serve as three-dimensional skeletal materials that provide a good matrix for cell culture and proliferation in vitro [ 128 ], but they are also widely used in bone defect repair, wound healing, and nerve repair owing to their ability to precisely adjust the composition and structure within the hydrogel to guide cell differentiation and promote neoplastic tissue growth, as well as their ability to deliver regenerative medicines to the tissues [ 129 , 130 ]. Biomaterials used to promote tissue regeneration must have strong mechanical strength to organize various living cells and functional factors in three dimensions. Specifically, DNA hydrogels containing bone marrow stem cells (BMSC) are injected directly into cartilage defects to construct cartilage-like organs. The DNA hydrogels provide a three-dimensional network scaffold that is comparable to the extracellular matrix (ECM) of cartilage, guiding and supporting the proliferation of chondrocytes while maintaining their physiological functions [ 131 ]. Hybridized DNA hydrogels have superior mechanical properties compared to pure DNA hydrogels [ 132 ]. In 2023, Zhou et al. [ 133 ] pioneered the construction of a dual-network DNA-silk fibronectin (SF) hydrogel. The first network consists of DNA through base complementary pairing to form a constraining supramolecular network, and SF molecules can form a second network structure through enzymatic cross-linking that acts as a molecular scaffold for DNA. The moderate surface stiffness of dual-network DNA-SF hydrogels is also able to promote collagen expression in the extracellular matrix and induce chondrogenic BMSC differentiation, synergistically promoting cartilage regeneration and repair. Compared to discrete DNA nanostructures, DNA-SF hydrogels maintain a more localized effect due to their polymerization and confinement to a defective region. Furthermore, in addition to excellent mechanical strength, for hydrogel dressings applied to wounds, good fit and gripping power are essential, and Ye et al. [ 134 ]. prepared a DNA hydrogel dressing with good fluid absorption and stable adhesion (Fig.  7 A), and on mouse liver, the DNA hydrogel quickly adhered to the wound and stopped bleeding. Similarly, Zhou et al. [ 135 ] designed biomimetic macro deformed DNA gel microneedles. Unlike the traditional MN array structure, MNs were designed to approximate a crab-claw-like structure and a shark microgroove structure, which improved the stability and gripping power (Fig.  7 B). The hydrogel adheres to the joint and remains stable after repeated deformation, which improves the comfort of patients with joint wounds. In conclusion, this DNA gel MN one-piece dressing is stable enough to regulate the wound microenvironment and promote high-quality wound healing. ( A ) Adhesive DNA Hydrogel Band-Aid for Hemostasis. Adapted reprinted with permission from Ref [ 134 ]. Copyright 2024, Springer Nature Ltd. ( B ) Dual bionic deformable DNA hydrogel microneedle-guided tissue regeneration in diabetic ulcer wounds, mechanical strength testing, capsule adhesion effect on joints and deformation testing. Adapted reprinted with permission from Ref [ 135 ]. Copyright 2023, Wiley. ( C ) Highly permeable DNA hydrogel promotes spinal cord repair. SCI: Injury-only group Injury-only group. M: Hydrogel without NSCs group, N: NSCs without hydrogel group, NM: Hydrogel loaded NSCs group Hydrogel with NSCs group. Adapted reprinted with permission from Ref [ 136 ]. Copyright 2021, Wiley DNA hydrogels meet most of the requirements for an ideal material for transplantation of neural stem cells (NSCs). Sequentially engineered DNA hydrogels serve as carriers for NSC transplants, and their permeability ensures the successful diffusion of nutrients and molecular signals into the tissue. In 2021, Liu et al. [ 136 ] reported a DNA supramolecular hydrogel with high permeability to repair spinal cord transection injury. The hydrogel was self-assembled from DNA double strands to host homologous neural stem cells, and the spacing of cross-linking sites between DNA double strands in the hydrogel was designed to be 20 nm (60 bp), which avoids the formation of small lattices preventing permeation and ensures that the hydrogel is useful for the rapid diffusion of neuronally relevant growth factors and other nutrients in the tissues. Injecting DNA hydrogels and NSCs into a surgically formed murine spinal cord defect model filled most of the defective cavities after eight weeks (Fig.  7 C). Furthermore, motor-evoked potential signals were detected in the hind limbs of the mice, suggesting that the hydrogel had successfully improved the regeneration of the tissues and recovered function. Also using DNA hydrogels to carry cells for tissue repair, Zhou et al. [ 137 ] designed a dual network hydrogel microsphere structure based on photocrosslinking. Compared to conventional block hydrogels, hydrogel microspheres are more efficient in solute diffusion and more conducive to promoting enhanced oxygen and nutrient exchange to enhance cell activity and differentiation potential. As shown in Fig.  8 A, the mixed hybrid filipin protein-DNA droplets were in the aqueous phase, which were encapsulated by the outer oil phase to form microspheres, and hydrogel microspheres with a bilayer network structure were formed under UV irradiation. Microspheres with a large specific surface area can enhance the cell diffusion rate and cell-cell interaction, which promotes the proliferation of attached cells and facilitates the construction of cartilage-like organs. Wound dressings stop bleeding, maintain moisture, prevent bacterial invasion, and promote wound healing. Multi-functional DNA hydrogels are considered an ideal skin substitute and wound dressing due to their excellent biodegradability, tissue adhesion, and capacity to carry a range of big and small molecule medications [ 138 ]. Antimicrobial peptides have attracted interest as structural templates for novel anti-infective drugs due to their low susceptibility to multidrug resistance mechanisms [ 139 ]. For example, the electrostatic interaction of a polyanionic DNA backbone and a cationic antimicrobial peptide (AMP) was used to form a physically cross-linked DNA hydrogel network. The release of the antimicrobial L12 peptide is modulated in the presence of DNA enzymes [ 140 ]. This DNA hydrogel loaded with antimicrobial L12 peptide showed significant efficacy in Staphylococcus aureus -infected porcine wounds. Treating chronic wounds, especially diabetic infected wounds, is one of the key problems to be solved in regenerative medicine and since the microenvironment of diabetic wounds is complex, physicians usually choose DNA hydrogels with various functions such as anti-inflammatory, antioxidant, and pro-angiogenic. For example, a physically crosslinked DNA hydrogel that ensures cytokine bioactivity and sustained release has been developed [ 141 ]. As shown in Fig.  8 B, an equiproportional mix of IL-33 and DNA monomers ensured uniform encapsulation of the cytokine. Under physiological conditions, this DNA gel sustains the release of IL-33 in the wound for at least seven days and is effective in inducing the local accumulation of immune cells to promote localized wound inflammation to subside. In addition, the DNA strand eliminated excess reactive oxygen species (ROS), which affect diabetic wound healing. Based on the same strategy, Yang et al. [ 142 ] designed a novel injectable DNA hydrogel dressing. Diversifying from the commonly used antimicrobial AgNCs, they physically encapsulated magnesium pyrophosphate crystals as an antimicrobial functional unit in a DNA polymer network, which slowly releases magnesium ions to promote wound angiogenesis in the wound microenvironment. The anti-inflammatory and antioxidant curcumin and antibiotic ciprofloxacin (CIP) were added, and the DNA hydrogel under the synergistic effect of the three showed excellent ROS scavenging and anti-inflammatory and antibacterial abilities, which effectively accelerated the healing of the infected wounds of diabetic patients. ( A ) Schematic diagram of synthesis and promotion of cartilage repair by RGD-SF-DNA hydrogel microspheres. Sham group: positive control, Control group: untreated group, RSD-MS group: hydrogel with RSD-MSs only, COP group: hydrogel with COPs only. Adapted reprinted with permission from Ref [ 137 ]. Copyright 2024, Elsevier Ltd. ( B ) Antioxidant DNA Hydrogels Deliver Cytokines to Promote Diabetic Wound Healing Hydrogel. Electron microscopy images and schematic diagrams of ROS scavenging, live/dead staining images of human keratinocytes cells cultured for 24 h in DNA hydrogel and no DNA hydrogel. Adapted reprinted with permission from Ref [ 141 ]. Copyright 2022, Wiley Bio-3D printing Using biomaterials to build three-dimensional tissue structures through interactions between cells and materials, 3D bioprinting is a sensitive tissue creation technique that may be used to repair damaged tissue and restore function. Precisely designed DNA hydrogels can meet the needs of 3D bioprinting. 3D bioprinting has gained attention for its ability to accurately print complex structures; however, selecting the correct scaffold material as bioink is the key to bioprinting. Hydrogels as scaffold materials have been widely reported due to their similarity to the natural extracellular matrix. However, the use of various natural products as scaffold materials for bioprinting has many drawbacks, such as high temperature-induced deformation of hydrogel formation and lack of responsiveness and customizability. Synthetic polymers in turn reduce the biodegradability and biocompatibility of hydrogels. Combining the concept of dynamic DNA nanotechnology with 3D bioprinting enables the production of hydrogel structures functionalized with DNA at the millimeter to centimeter scale [ 143 ]. Li et al. [ 144 ] printed peptide-DNA hydrogels using two bioinks with different compositions. As shown in Fig.  9 A, bioink A is a peptide backbone with five to six ssDNA motifs reconnected to create enough cross-linking sites. Bioink B is a double-stranded DNA (dsDNA) containing sticky ends that are complementary to the Bioink A ssDNA and act as DNA junctions. Once the printed droplets touch and mix, the two bioinks rapidly crosslink to form a supramolecular DNA hydrogel in less than a second. The hydrogels formed by this 3D printing are very rapid compared to hydrogel formation by manual mixing. The bioprinter can also be programmed to precisely control the position and distance of the printed droplets. This printed DNA hydrogel has borderless geometric homogeneity and maintains millimeter-scale shapes without collapsing, exhibiting good mechanical flexibility and healing properties. In addition, they added cells to the ink for testing, and unexpectedly, the specific viscosity and surface tension of the bio-ink not only met the requirements of the nozzle technology, but also the cells were able to remain stable in suspension and had high viability and normal biological functions. Therefore, with this bioprinting system, not only 3D patterns and structures of arbitrary scale and size can be constructed, but also long-term cell cultures are promising. Researchers developed a low-cost method for 3D bioprinting based on a commercially available extrusion printer [ 145 ], to develop a bioink covalently modified with DNA molecules. Agarose was modified with ssDNA strands to form the bioink, and DNA-functionalized hydrogels of various shapes were printed. Another advantage of 3D printing is the ability to precisely fabricate porous scaffolds with controllable shapes, and the printed structures can maintain millimeter-scale porous shapes without collapsing. Chen et al. [ 146 ] supramolecularly co-assembled amyloid fibrillar proteins (AFs), clay nanosheets, and DNA chains to develop a hybrid DNA hydrogel (DAC) with 3D printing properties. DNA hydrogels are formed by electrostatic interactions between the positively charged amyloid fibrillar protein and the negatively charged DNA chains and clay nanosheets. A patterned macroporous structure was generated by a regular arrangement of hydrogel filaments in which a significant number of interconnecting pores were evenly distributed owing to freeze-drying, as seen in Fig.  9 B for the DAC hydrogel scaffolds made in various forms by 3D printing. Additionally, the 3D printed DAC hydrogel scaffold exhibited potential for withstanding the somewhat acidic environment of in vivo wounds since it remained stable in various pH settings without experiencing appreciable swelling or disintegration. Cunniffe et al. [ 147 ] designed a novel printing ink consisting of alginate and nanohydroxyapatite (nHA) complexed with plasmid DNA (pDNA). Bone marrow mesenchymal stem cells were placed in the ink, which can form a stable network structure to provide mechanical stability to the constructs during printing. In addition, the four different extragenic genes used in the printing process transfect reporter and therapeutic genes onto the MSCs, thereby allowing the bone marrow MSCs to differentiate into osteoblasts and promoting osteogenic differentiation. Thus, this hydrogel induces MSC differentiation and bone regeneration through bioprinting. In addition, the construct containing nHA-pDNA showed enhanced osteogenic capacity compared to the construct containing nHA alone. DNA hydrogels are ideal materials for bioprinting. Most of the existing DNA hydrogels for 3D bioprinting are mainly based on the bottom-up strategy, i.e., the preparation of hydrogels that can be rapidly gelatinized and then made into bioinks for printing. When DNA hydrogels are used as bioinks for 3D printing, complex and customized skeletal structures can be fabricated through sophisticated instrumentation, which can improve the efficiency of DNA hydrogel preparation. Notably, tissue structures printed using hybrid or pure DNA bioinks may exhibit reduced levels of inflammation or foreign body reactions, and their biocompatibility may be somewhat weakened [ 139 ]. Challenges remain that need to be resolved in order to maximize the printing process. For example, DNA hydrogels for fabricating tissues and organs require large amounts of polymers and are costly [ 148 ]. Moreover, the mechanical properties and stability of printed DNA hydrogels are poor, which makes it difficult to guarantee the long-term activity of loaded cells in tissue engineering applications. In summary, DNA hydrogel 3D printing presents great potential and requires additional in-depth studies. ( A ) 3D bioprinting of peptide-DNA hydrogels. Adapted reprinted with permission from Ref [ 144 ]. Copyright 2023, American Chemical Society. ( B ) 3D printed dual nanoengineered dynamic DNA hydrogel 3D printing technique to make symmetric DAC1.0 hydrogel scaffolds. Adapted reprinted with permission from Ref [ 146 ]. Copyright 2015, Wiley Cell culture and capture A vital component of the cellular environment, the ECM, is a dynamic network of collagen, glycoproteins, enzymes, and other macromolecules that regulates cell activity [ 149 ]. Due to their high water content, DNA hydrogels resemble the ECM in that they exhibit gel-like characteristics. Additionally, their porous structure facilitates the flow of nutrients and metabolic wastes, hence promoting high cellular viability. High cell viability is maintained by the porous structure, which permits the flow of nutrients and metabolic wastes. DNA hydrogels are therefore ideally suited for significant cell culture-based applications. Introducing a sequence of materials into DNA hydrogels increases their versatility in cell culture. DNA sequences bind to specific cellular receptors in a targeted manner, thus ensuring cell immobilization and cell culture. However, the rigidity of the DNA structure reduces the mechanical properties of the hydrogel, which is prone to collapse when insufficient support is provided. Therefore, researchers have developed multifunctional DNA nanostructures for assisting target-specific adhesion and cell proliferation in cell cultures [ 150 ]. As shown in Fig.  10 A, four complementary ssDNA sequences were first functionalized with acrylates (as photo-crosslinkers) or peptidomers (as cell adhesion molecules), respectively, to prepare multifunctional nanostructures of X-DNA, and hybrid hydrogels formed by photo-crosslinking rapidly under mild reaction conditions. Various types of functionalized X-DNA (AptX-DNA) can be synthesized by controlling the ratio of photocrosslinker to aptamer added to X-DNA. Furthermore, aptamers were inserted into the branched strands to optimize the cell adhesion of X-DNA and promote cell proliferation. In the same year, another study introduced polyacrylamide hydrogels as additional networks. Gao et al. [ 151 ] developed a new strategy for constructing DNA-polyacrylamide (PAAm) hybridization hydrogel preparation. The dual network formed by the DNA strands and polyacrylamide improves the tensile and shear strength of the hydrogel and ensures that it remains stable during performing immunostaining and cellular imaging to visualize the cellular behaviors and functions in a 3D environment. Traditional 2D cell culture systems performed on planar scaffolds lack cell-cell and cell-environment interactions [ 152 , 153 , 154 ]. However, hydrogels with a jelly-like texture with high water content not only provide effective physiological and structural support for 3D cell growth, but can also be tuned with biochemical and physical properties to mimic the extracellular matrix, which shows great potential in 3D cell culture [ 155 ]. However, nucleases in the culture medium degrade the hydrogel structure, leading to a significant reduction in the shelf life of DNA hydrogels. Yao et al. [ 156 ] proposed a novel strategy for extending the validity period of DNA hydrogels. Poly (L-lysine) (PLL) was used as a cross-linking agent to connect single-stranded DNA integrated with an aptamer for the rapid assembly of the hydrogel network (Fig.  10 B). PLL served as a protective coating to increase the resistance between the nuclease and the phosphodiester bond, effectively preventing nuclease damage to the DNA hydrogel network. After 15 days of cell culture, cells encapsulated in the hydrogel were able to proliferate and eventually form cell spheroids, indicating that the coating improves the stability of the hydrogel structure without affecting the ability of the aptamer to target and recognize cells. ( A ) Functionalized Aptamer-DNA nanostructures for enhanced cell culture. Adapted reprinted with permission from Ref [ 150 ]. Copyright 2021, American Chemical Society. ( B ) DNA/polylysine hydrogels for three-dimensional cell culture. Adapted reprinted with permission from Ref [ 156 ]. Copyright 2024, Wiley In addition to cell culture, DNA hydrogels can capture specific cells efficiently while maintaining high cell viability. Yao et al. [ 157 ] introduced a DNA hydrogel network for the efficient capture of bone marrow MSCs. As shown in Fig.  11 A, in the RCA-based synthesis of two complementary DNA long strands, they were mixed and entwined to form a hydrogel. The sequence of one of the DNA strands contains an aptamer with high affinity to the special protein on the membrane of BMSCs, which is used to specifically capture BMSCs from bone marrow, whereafter the DNA strand is deconstructed by the addition of nuclease to release the captured cells. Distinguishing from the use of long DNA strands to construct a web to encapsulate cells, Tang et al. [ 51 ] reported another hydrogel formed using electrostatic attraction and interfacial assembly of long DNA strands and UCNPs. The addition of NPs caused rapid hydrogel formation from the mixed solution, which selectively captured the target cells. Tumor cell detection and consolidation therapy are important for patients with cancer after surgery. If live circulating tumor cells (CTCs) are accurately and efficiently isolated from peripheral blood and monitored in real time, tumor recurrence can be sensitively detected, which is important for the prevention of secondary metastasis. The programmability of a DNA molecule allows DNA networks to have the desired structure and specific functions. In 2020, Li et al. [ 158 ] synthesized physically cross-linked DNA hybrid DNA hydrogels containing ATP-responsive aptamers (Fig.  11 B). When the aptamer binds to epithelial adhesion molecules on the surface of tumor circulating cells, it triggers the formation of porous DNA hydrogels. Using this system, blood from cancer patients successfully encapsulated and released CTCs, identifying as few as 10 tumor cells in 2 µL of whole blood, and the special composition of the DNA hydrogel can both capture and kill cells. It is worth mentioning that, also utilizing aptamers for cell-targeted capture, Mu et al. [ 159 ] designed an anisotropic DNA hydrogel. This specially structured hydrogel consists of unidirectional pore channels, and compared with the control DNA hydrogel without macroporous channels, the DNA hydrogel with macroporous channels exhibits more efficient cell capture. Recently, Wang et al. [ 160 ] developed a local photodynamic immunomodulatory DNA hydrogel. One ssDNA strand contained the complementary sequence of the immune adjuvant CpG and the PDL1 aptamer, and the other contained the ATP sensor. When the PDL1 aptamer binds to the PDL1 protein on the surface of the tumor cell, the tumor cell is anchored and aggregates, the local ATP concentration increases, the ATP sensor emits fluorescent signals, and the photosensitizer added in advance induces the hydrogel to decompose by local laser irradiation, releasing CpG and PDL1 aptamers, and inducing the immune response to kill the tumor cells. The more PDL1 aptamers are released from the hydrogel, the more tumor cells are captured, the stronger the fluorescence signal is, and the more hydrogel is released from the hydrogel decomposition, forming a benign signal amplification cycle. However, the hydrogel is only able to monitor part of the tumor site, the tumor may still recur, and the stability of the hydrogel in the body remains to be considered. In 2021, Jiang et al. [ 161 ] established a Zn 2+ -dependent DNA enzyme responsive DNA hydrogel (Fig.  11 C). Similar to the above strategy, the hydrogel is constructed by intertwining and hybridizing two polymerized DNA strands (R1 and R2), the R1 strand containing the aptamer sequence and DNAzyme sequence, and the R2 strand containing the corresponding DNAzyme substrate sequence. When R2 is added to the R1 solution, R2 is entangled with the R1 strand of the target cell already captured in the solution to form a DNAzyme hydrogel, and the target cell is separated by solution-gel conversion. The DNAase hydrogel disassembles and releases the captured cells when triggered by Zn 2+ . The entire process of Zn 2+ has minimal cytotoxicity, and the capture and release of the DNA hydrogel are performed in mild conditions, which is expected to be suitable for the use of clinical samples. ( A ) Physical crosslinked DNA networks for stem cell harvesting. Adapted reprinted with permission from Ref [ 157 ]. Copyright 2020, American Chemical Society. ( B ) Encapsulation of live tumor cells in blood using a hydrogel that reacts with hybridization chains. Adapted reprinted with permission from Ref. The process of ultra-long DNA strands to obtain three-dimensional DNA networks as well as the process of capturing, encapsulating and releasing BMSCs [ 158 ]. Copyright 2021, American Chemical Society. ( C ) DNA enzyme-triggered solution-gel-solution transition of the hydrogel enriches target cells. Adapted reprinted with permission from Ref [ 161 ]. Copyright 2020, Springer Nature Ltd Conclusion and prospects Nowadays, many ingenious strategies for the preparation of DNA hydrogels have been developed through various precise structural designs. Pure DNA hydrogel network gels based on hydrogen bonding of base complementary pairing usually have stable and controllable structures. Hydrogels for nucleic acid amplification to obtain ultra-long DNA strands that are then physically wound is an easy alternative. Gradually, researchers have added single or multiple functional materials with excellent properties to the hydrogels, such as functional DNA groups, natural and synthetic polymers, and various new nanomaterials, as well as DNA as a crosslinking agent to tightly connect various functional materials to each other, so as to make them have the required optical, electronic, and other physical properties, to realize different DNA hydrogel functions. The mechanical, optical, and encapsulating capabilities of these functionalized DNA hydrogels are used in the biosensing area to meet the requirements of high sensitivity and accuracy in sensing detection. To achieve the accurate distribution of loaded pharmaceuticals using DNA hydrogels, we concentrated on discussing functional DNA hydrogels with high mechanical capabilities and stimulation response properties for use in targeted therapy and drug delivery. DNA hydrogels must have strong molecular permeability, thixotropy, self-healing, and antibacterial qualities in tissue engineering and cell culture applications to adjust to the right environments for cell growth and differentiation. However, DNA hydrogels have many drawbacks. For example, as the preparation cost of DNA hydrogels is still too high to meet the requirements for scaling up production, it is hoped that simpler synthesis methods can be developed to reduce costs, and the mechanical properties of DNA hydrogels need to be improved, especially as biomedical materials used in clinical applications should have repair and stretching capabilities similar to human tissues, while the biocompatibility of the hydrogel material should be ensured; therefore, programmable DNA scaffolds with adjustable stiffness are ideal. When testing metrics of biological systems, it may be possible to integrate DNA hydrogel assay designs with mobile apps to allow for more intuitive real-time monitoring, thus developing more individualized treatments tailored to the patient. Furthermore, the development of specific DNA sequences in hydrogels for binding to biomolecules of interest is important for guiding both cell behavior and tissue engineering. In addition, many hybrid DNA hydrogels contain other materials as structural scaffolds, and when these hydrogels are used in the in vivo environment, we have to consider the toxic effects and biodegradability of the materials involved. We can use more biocompatible materials such as exosomes, herbal molecules with anti-inflammatory properties. Extensive studies using animal models and perhaps clinical trials must be conducted going forward. To address the remaining issues in hydrogel application, the properties of DNA hydrogels should be continuously optimized and expanded. 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Download references Acknowledgements This work was supported by Hunan Provincial Health Commission Scientific Research Project, China (C202304106744), and Zhejiang Shuren University research project (2023R053 and 2023KJ237). Author information Authors and affiliations. Department of Plastic and Aesthetic (Burn) Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China Rui Wu, Pu Yang, Naisi Shen, Anqi Yang, Xiangjun Liu, Yikun Ju & Bairong Fang Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences School of Basic Medicine, Peking Union Medical College, Beijing, 100000, China Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China You can also search for this author in PubMed   Google Scholar Contributions R.W. wrote and revised the main manuscript. W.L. conceived the project. P.Y., N.S., A.Y., X.L., Y.J. reviewed the manuscript. L.L. and BF conceived and designed the manuscript. All authors read and approved the final manuscript. Corresponding authors Correspondence to Lanjie Lei or Bairong Fang . Ethics declarations Ethics approval and consent to participate. Not applicable. Consent for publication We give our consent for the manuscript to be published in Journal of Nanobiotechnology. Competing interests The authors declare no competing interests. Additional information Publisher’s note. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Provided by the Springer Nature SharedIt content-sharing initiative DNA hydrogels Self-assembly Crosslinking Mechanical-properties Biomedical application Journal of Nanobiotechnology ISSN: 1477-3155 Submission enquiries: [email protected] 408 IMAGES Semi-Conservative Replication Diagram of the Meselson Stahl Experiment DNA Poster Meselson Stahl Experiment PPT The Most Beautiful Experiment in Biology Meselson and Stahl VIDEO L-08 Получение этилена и изучение его свойств. Semiconservative DNA Replication Class12th,NEET MESELSON-STAHL EXPERIMENT [ dna recombination ] || BY PHANINDRA GUPTHA || Meselson and stahl’s experiments || Question || Molecular basis of inheritance || NEET Molecular basis of inheritance COMMENTS Meselson-Stahl experiment The Meselson-Stahl experiment is an experiment by Matthew Meselson and Franklin Stahl in 1958 which supported Watson and Crick's hypothesis that DNA replication was semiconservative.In semiconservative replication, when the double-stranded DNA helix is replicated, each of the two new double-stranded DNA helices consisted of one strand from the original helix and one newly synthesized. The Meselson-Stahl Experiment (1957-1958), by Matthew Meselson and In an experiment later named for them, Matthew Stanley Meselson and Franklin William Stahl in the US demonstrated during the 1950s the semi-conservative replication of DNA, such that each daughter DNA molecule contains one new daughter subunit and one subunit conserved from the parental DNA molecule. The researchers conducted the experiment at California Institute of Technology (Caltech) in ... How DNA Replicates Video 1 Matt Meselson describes his experiment with Frank Stahl on DNA replication. The genetic material of eukaryotic cells is organized in the form of chromosomes, each a single linear, double-stranded DNA molecule (Figure 1). Most prokaryotes (bacteria) have a single, circular chromosome (Figure 1). All forms of life must replicate their DNA ... Semi-Conservative DNA Replication: Meselson and Stahl However, it took several years of subsequent study, including a classic 1958 experiment by American geneticists Matthew Meselson and Franklin Stahl, before the exact relationship between DNA ... Meselson-Stahl Experiment DNA containing a mixture of 15 N and 14 N ends up in an intermediate position between the two extremes. By spinning DNA extracted at different times during the experiment, Meselson and Stahl were able to see how new and old DNA interacted during each round of replication. The beauty of this experiment was that it allowed them to distinguish ... Khan Academy Khanmigo is now free for all US educators! Plan lessons, develop exit tickets, and so much more with our AI teaching assistant. Classics: Meselson and Stahl: The art of DNA replication Matthew Meselson and Franklin Stahl's experiments on the replication of DNA, published in PNAS in 1958 ( 2 ), helped cement the concept of the double helix. Meselson, a graduate student, and Stahl, a postdoctoral researcher, both at the California Institute of Technology (Pasadena), gave validity to a model that many scientists saw as ... Meselson, Stahl and the Replication of DNA: A History of ... Nature Medicine - Meselson, Stahl and the Replication of DNA: A History of "The Most Beautiful Experiment in Biology" Meselson and Stahl: The art of DNA replication Matthew Meselson and Franklin Stahl's experiments on the replication of DNA, published in PNAS in 1958 ( 2), helped cement the concept of the double helix.Meselson, a graduate student, and Stahl, a postdoctoral researcher, both at the California Institute of Technology (Pasadena), gave validity to a model that many scientists saw as speculation: how two intertwined and tangled strands of a ... The Meselson And Stahl Experiment on DNA Replication Experiment. 15 N (heavy) and 14 N (normal) are two isotopes of nitrogen, which can be distinguished based on their densities by centrifugation in Ca,esium chloride (CsCl). Meselson and Stahl cultured E.coli in a medium constituting 15 NH 4 Cl over many generations. As a result, 15 N was integrated into the bacterial DNA. Later, they revised the 15 NH 4 Cl medium to normal 14 NH 4 Cl. Meselson and Stahl Experiment Meselson and Stahl experiment gave the experimental evidence of DNA replication to be semi-conservative type.It was introduced by the Matthew Meselson and Franklin Stahl in the year 1958.Matthew Meselson and Franklin Stahl have used E.coli as the "Model organism" to explain the semiconservative mode of replication. There are three modes of replication introduced during the 1950s like ... The Most Beautiful Experiment: Meselson and Stahl • iBiology Matt Meselson and Frank Stahl were in their mid-20s when they performed what is now recognized as one of the most beautiful experiments in modern biology. In this short film, Matt and Frank share how they devised the groundbreaking experiment that proved semiconservative DNA replication, what it was like to see the results for the first time ... The Meselson Stahl Experiment. Author: David Sadava. The Meselson Stahl Experiment. A centrifuge was used to separate DNA molecules labeled with isotopes of different densities. This experiment revealed a pattern that supports ... The Meselson-Stahl Experiment: "the Most Beautiful Experiment in We begin with the theoretical and experimental background that provided the motivation and context for the Meselson-Stahl experiment. Footnote 1 In 1953 Francis Crick and James Watson proposed a three-dimensional structure for deoxyribonucleic acid (DNA): two polynucleotide chains helically wound about a common axis (Watson and Crick 1958).This was the famous "Double Helix" where the ... Meselson-Stahl Experiment Paul Andersen explains how the Meselson-Stahl experiment was used to prove that DNA copied itself through a semi-conservative process. They grew E. coli in ... The Most Beautiful Experiment: Meselson and Stahl Matt Meselson and Frank Stahl share the story of their groundbreaking experiment from 1958 that definitively showed semiconservative DNA replication.Matt Mes... An Elegant Experiment to Test the Process of DNA Replication Testing predictions is a major part of scientific research, and a key component of many classic experiments. This module explores the research methods used by Meselson and Stahl in their ingenious 1958 experiment showing how DNA replicates. The module highlights the power of simplicity in what has been called the most beautiful experiment in biology. Density matters: The semiconservative replication of DNA A comprehensive historical description of the collaboration between Meselson and Stahl, the milieu in which they worked, and their remarkable path to success was prepared by the late Frederic Lawrence Holmes and titled Meselson, Stahl, and the Replication of DNA: A History of "the Most Beautiful Experiment in Biology" ( 2).This account highlights the personalized side of the story and ... Meselson and Stahl Experiment Meselson and Stahl performed the density-gradient centrifugation of DNA in a solution of calcium chloride. 5. Starting with 15 N 15 N (heavy) DNA, and after ONE generation in the 14 N medium, E. coli cells subjected to density-gradient centrifugation will produce a single band of DNA with medium density. Scientists unlock the secrets of how a key protein converts DNA into RNA The team is already working on time-resolved experiments to capture the real-time dynamics of the polymerase's trigger loop as it interacts with nucleotides with the hopes of unraveling the ... DNA hydrogels and their derivatives in biomedical engineering Deoxyribonucleotide (DNA) is uniquely programmable and biocompatible, and exhibits unique appeal as a biomaterial as it can be precisely designed and programmed to construct arbitrary shapes. DNA hydrogels are polymer networks comprising cross-linked DNA strands. As DNA hydrogels present programmability, biocompatibility, and stimulus responsiveness, they are extensively explored in the field ... essay homework paper phd project resume review speech thesis