meselson and stahl experiment procedure

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Biology archive

Course: biology archive > unit 17.

DNA replication and RNA transcription and translation
Leading and lagging strands in DNA replication
Speed and precision of DNA replication
Molecular structure of DNA
Molecular mechanism of DNA replication

Mode of DNA replication: Meselson-Stahl experiment

DNA proofreading and repair
Telomeres and telomerase
DNA replication

Key points:

There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive.
The semi-conservative model, in which each strand of DNA serves as a template to make a new, complementary strand, seemed most likely based on DNA's structure.
The models were tested by Meselson and Stahl, who labeled the DNA of bacteria across generations using isotopes of nitrogen.
From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that DNA is replicated semi-conservatively.

Mode of DNA replication

The three models for dna replication.

Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).
Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

Meselson and Stahl cracked the puzzle

The meselson-stahl experiment, results of the experiment, generation 0, generation 1, generation 2, generations 3 and 4, attribution:, works cited:.

Watson, J. D. and Crick, F. H. C. (1953). A structure for deoxyribose nucleic acid. Nature , 171 (4356), 737-738. Retrieved from http://www.nature.com/nature/dna50/watsoncrick.pdf .
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). The basic principle: Base pairing to a template strand. In Campbell biology (10th ed.). San Francisco, CA: Pearson, 318-319.
American Institute of Biological Sciences. (2003). Biology's most beautiful. http://www.aibs.org/about-aibs/030712_take_the_bioscience_challenge.html .
Watson, J. D., and Crick, F. H. C. (1953). Genetical implications of the structure of deoxyribonucleic acid. Nature , 171 , 740-741.
Davis, T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS , 101 (52), 17895-17896. http://dx.doi.org/10.1073/pnas.0407540101 .

References:

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

Illustration of the Meselson-Stahl Experiment

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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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:

predictions for f1 gen made by meselson and stahl

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.

predictions for f2 gen made by meselson and stahl

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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Semi-Conservative DNA Replication: Meselson and Stahl

This structure has novel features which are of considerable biological interest . . . It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material . —Watson & Crick (1953)

Perhaps the most significant aspect of Watson and Crick's discovery of DNA structure was not that it provided scientists with a three-dimensional model of this molecule , but rather that this structure seemed to reveal the way in which DNA was replicated. As noted in their 1953 paper, Watson and Crick strongly suspected that the specific base pairings within the DNA double helix existed in order to ensure a controlled system of DNA replication . 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 structure and replication was understood.

Three Proposed Models for DNA Replication

Replication is the process by which a cell copies its DNA prior to division. In humans, for example, each parent cell must copy its entire six billion base pairs of DNA before undergoing mitosis . The molecular details of DNA replication are described elsewhere, and they were not known until some time after Watson and Crick's discovery. In fact, before such details could be determined, scientists were faced with a more fundamental research concern. Specifically, they wanted to know the overall nature of the process by which DNA replication occurs.

Defining the Models

Semiconservative replication was not the only model of DNA replication proposed during the mid-1950s, however. In fact, two other prominent hypotheses were put also forth: conservative replication and dispersive replication. According to the conservative replication model, the entire original DNA double helix serves as a template for a new double helix, such that each round of cell division produces one daughter cell with a completely new DNA double helix and another daughter cell with a completely intact old (or original) DNA double helix. On the other hand, in the dispersive replication model, the original DNA double helix breaks apart into fragments, and each fragment then serves as a template for a new DNA fragment. As a result, every cell division produces two cells with varying amounts of old and new DNA (Figure 1).

Making Predictions Based on the Models

Meselson and stahl’s elegant experiment.

The duo thus began their experiment by choosing two isotopes of nitrogen—the common and lighter 14 N, and the rare and heavier 15 N (so-called "heavy" nitrogen)—as their labels and a technique known as cesium chloride (CsCl) equilibrium density gradient centrifugation as their sedimentation method. Meselson and Stahl opted for nitrogen because it is an essential chemical component of DNA; therefore, every time a cell divides and its DNA replicates, it incorporates new N atoms into the DNA of either one or both of its two daughter cells, depending on which model was correct. "If several different density species of DNA are present," they predicted, "each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way, DNA labeled with heavy nitrogen ( 15 N) may be resolved from unlabeled DNA" (Meselson & Stahl, 1958).

The scientists then continued their experiment by growing a culture of E. coli bacteria in a medium that had the heavier 15 N (in the form of 15 N-labeled ammonium chloride) as its only source of nitrogen. In fact, they did this for 14 bacterial generations, which was long enough to create a population of bacterial cells that contained only the heavier isotope (all the original 14 N-containing cells had died by then). Next, they changed the medium to one containing only 14 N-labeled ammonium salts as the sole nitrogen source. So, from that point onward, every new strand of DNA would be built with 14 N rather than 15 N.

Just prior to the addition of 14 N and periodically thereafter, as the bacterial cells grew and replicated, Meselson and Stahl sampled DNA for use in equilibrium density gradient centrifugation to determine how much 15 N (from the original or old DNA) versus 14 N (from the new DNA) was present. For the centrifugation procedure, they mixed the DNA samples with a solution of cesium chloride and then centrifuged the samples for enough time to allow the heavier 15 N and lighter 14 N DNA to migrate to different positions in the centrifuge tube.

By way of centrifugation, the scientists found that DNA composed entirely of 15 N-labeled DNA (i.e., DNA collected just prior to changing the culture from one containing only 15 N to one containing only 14 N) formed a single distinct band, because both of its strands were made entirely in the "heavy" nitrogen medium. Following a single round of replication, the DNA again formed a single distinct band, but the band was located in a different position along the centrifugation gradient. Specifically, it was found midway between where all the 15 N and all the 14 N DNA would have migrated—in other words, halfway between "heavy" and "light" (Figure 2). Based on these findings, the scientists were immediately able to exclude the conservative model of replication as a possibility. After all, if DNA replicated conservatively, there should have been two distinct bands after a single round of replication; half of the new DNA would have migrated to the same position as it did before the culture was transferred to the 14 N-containing medium (i.e., to the "heavy" position), and only the other half would have migrated to the new position (i.e., to the "light" position). That left the scientists with only two options: either DNA replicated semiconservatively, as Watson and Crick had predicted, or it replicated dispersively.

Straight or Circular?

Following publication of Meselson and Stahl's results, many scientists confirmed that semiconservative replication was the rule, not just in E. coli , but in every other species studied as well. To date, no one has found any evidence for either conservative or dispersive DNA replication. Scientists have found, however, that semiconservative replication can occur in different ways—for example, it may proceed in either a circular or a linear fashion, depending on chromosome shape.

In fact, in the early 1960s, English molecular biologist John Cairns performed another remarkably elegant experiment to demonstrate that E. coli and other bacteria with circular chromosomes undergo what he termed " theta replication ," because the structure generated resembles the Greek letter theta (Θ). Specifically, Cairns grew E. coli bacteria in the presence of radioactive nucleotides such that, after replication, each new DNA molecule had one radioactive (hot) strand and one nonradioactive strand. He then isolated the newly replicated DNA and used it to produce an electron micrograph image of the Θ-shaped replication process (Figure 3; Cairns, 1961).

References and Recommended Reading

Cairns, J. The bacterial chromosome and its manner of replication as seen by autoradiography. Journal of Molecular Biology 6 , 208–213 (1961)

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

Watson, J. D., & Crick, F. H. C. A structure for deoxyribose nucleic acid. Nature 171 , 737–738 (1953) ( link to article ).

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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.

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Proc Natl Acad Sci U S A
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.”

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.

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The Most Beautiful Experiment

Matt Meselson and Frank Stahl share the story of their groundbreaking experiment

About the Film

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.

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Frank Stahl, Ph.D.: John Cairns said on the telephone in a very excited voice, he had just read Mendel’s papers. [Imitating Cairns] “And you know they are the most beautiful experiments in Biology.” And I gasped and I said, “John, John, you can’t say that. You said the Meselson-Stahl experiment was the most beautiful experiment in Biology.” [Imitating Cairns] “Oh, did I? Well, I was wrong.”

Matt Meselson, Ph.D.: Watson and Crick didn’t make a discovery. They proposed a model. There are those who believed this model must be true because it was so beautiful. And there were those who believed it must be wrong because biology is complicated. And this model is too simple to be right. Would you say?

Stahl: Exactly, yes.

Meselson: But there was no experimental proof of it.

Stahl: They had a model which made a distinct prediction about how DNA replicates and it needed to be tested. And it’s fun to test the hypothesis. We agreed that we were going to work together to figure out whether or not it was right.

Meselson: When Frank and I showed semi-conservative replication, it wasn’t just a model, it was something real like that.

Stahl: After our experiment, it was now widely accepted that their model, the Watson-Crick model, is right. So it became the building block. You might say for all of biology.

Meselson: Yeah. From very early childhood, I mean, practically infancy, I loved science. I loved to put wires together to make little radios, which I could put under my pillow. So my parents wouldn’t know that I was listening to them all night long. And then I was very interested to know what makes life work. And there had been, I think, in the house, maybe even in my bedroom, that painting by Michelangelo, God up high and Adam below and they’re touching fingers. I don’t know if there is a spark. I don’t think there’s a spark in the picture, but I’m not sure. But to me that meant that life is somehow electrical. God is providing life through a spark. And for some reason that made me interested in electrochemistry.

Stahl: Unlike Matthew, I had no particularly strong interest in science as a youth. What was understood that when I graduated from high school, I would apply to the Naval Academy. That’s what my mother had in mind because she thought I would look good in dress whites. But then of course, World War II broke out. So that plan changed fast and she decided I should just go to college.

I think I was just too young to understand the courses in humanities. I hadn’t had enough life experience to get a grip on the questions they were even thinking about. Science on the other hand was concrete. Children can grasp science and among the sciences, biology was the most appealing. There, the fun was that you could figure out puzzles. That is, there was a rational, concrete, quantitative explanation for what you saw. You could reason backwards as to what must be going on. And that intrigued me enough to know that genetics was something perhaps I could do.

Meselson: I had the great, good luck to become Linus Pauling’s last graduate student. His daughter was having a party at their swimming pool and I’m in the water. And Pauling comes out, the world’s greatest chemist. I’m all naked, practically, in a bathing suit. And he’s all dressed up with a jacket and a vest and a neck tie. And he looked down at me, “Well, Matt, what are you gonna do next year?” And I had already signed up to go to the committee on mathematical biophysics and Linus just looked down at me and he said, “But Matt, that’s a lot of baloney. Come be my graduate student.”

And so if I hadn’t taken his course on the nature of the chemical bond, I would have had a very different life. I wouldn’t have met Frank, I wouldn’t be sitting here, that’s for sure. At the end of my PhD exam, as we were walking out of the little exam room, Linus Pauling turned to me and he said, “Matt, you’re very lucky you’re entering this field just at the right moment.”

Stahl: Yeah.

Meselson: At the very beginning. The first year of my being a graduate student at Caltech, I wanted to get into biology. I was a chemist and I thought the way to do that would be to study molecular structure. The only person who was looking at biology from that point of view, other than Linus Pauling himself, was Max Delbruck. He had a fearsome reputation. Nevertheless, I got up my courage and went to see him. He’s not a fearsome creature at all really. And the first thing he said was, “What do you think about these two papers from Watson and Crick?” I said, I’d never heard of them. I was still in the dark ages, and he yelled at me. He said, “Get out and don’t come back till you’ve read them.”

There were two separate ideas that came together. Crick’s idea about how the base pairs linked onto the chains and Jim’s idea about how the base pairs were structured. So there are four different building blocks in DNA, adenine, thymine, guanine, and cytosine.

Stahl: The surfaces of the G and the C are complementary to each other and of the A and T are complementary to each other so that they can fit together. The way fingers would fit into a glove. And importantly, when they put G opposite C, the distance of the outside was exactly the same as if they’d put A opposite T. No other combination would give such a regular structure. It was a gorgeous insight.

And then from that, they made a hypothesis about how DNA is replicated. It involved the two chains coming apart and each one acting as a template for the synthesis of a new chain on its surface. When it’s all done, here we have the two old chains, each one now associated with a brand new chain.

Meselson: What Watson and Crick proposed was enormous stimulus to experimentation.

Stahl: It was irresistibly beautiful.

Meselson: Irresistibly beautiful. Jim Watson was at Caltech the year after he and Francis published their papers. And so I got a chance to talk a lot with Jim then, and that coming summer he was going to go and teach the physiology course at Woods Hole.

Stahl: I was a graduate student at Rochester at the time. My chairman of the department who was also on my committee, said I had to take a course in physiology. And I said, the physiologist teacher here is a jerk. I’ll be damned if I’ll take his course. Well, send him to Woods Hole to take the physiology course there. And by serendipity, Jim Watson happened to be there with some kid named Meselson hanging along with him. We found that we had in fact deep, common interests.

Meselson: I realized this is a guy who’s really very smart and I can learn a lot from him.

Stahl: I remember a haze of beach parties, lectures that I slept through.

Meselson: Well it was a kind of paradise. The most interesting people in molecular biology. Most of them were there. So that’s how we met. And then it turns out Frank is coming that very September to Caltech.

Stahl: No, it would be a year from then I would come.

Meselson: Are you sure?

Stahl: Yep. I still hadn’t finished my thesis-

Meselson: So I had to wait for a whole year before I saw you again?

Stahl: That’s right. He said, “When you get to Caltech we’ll test Jim’s idea. What do you think about testing Jim’s idea of how DNA replicates?” And then he explained that to me, I’d already heard about it and he explained it to me and I absolutely- I committed, totally.

Meselson: And then when Frank finally got there and I wanted to start right away, he forbade it.

Interviewer: Why?

Meselson: He said it would be bad for my character to not complete my x-ray crystallography before starting something new. This tells you a lot about Frank’s character. With the Watson and Crick model, the underlying question of course was, was that really the right mechanism?

Stahl: The famous Max Delbruck said “No, no, no, no, that model can’t be right.” And he proposed a different model. As Delbruck put it forth, breaks are introduced in the parental molecule as it’s being replicated and then carefully sealed up in certain ways.

Others proposed one in which the original DNA molecule stays intact. And the new DNA molecule is made of all new DNA. So there were three targets out there that in principle could be distinguished, if you could trace the fate of the old chains, what becomes of the two old chains.

Meselson: And one step led to the next, really. I mean, the first idea was using density somehow, which is not a very good idea yet, except it leads you to the next one.

Stahl: Matt’s idea from the very beginning was that somehow stable isotopes could be used that would be incorporated into the DNA and impart upon the DNA, a different density.

Meselson: You grow bacteria in a medium, which instead of having this ordinary isotope of nitrogen, N14, you can buy nitrogen 15 ammonium chloride, the heavy kind. And if you grow the bacteria for a number of generations, you can be sure that essentially all of the DNA is labeled with heavy nitrogen, good. Now, we resuspend those cells in a medium that just has ordinary, nitrogen 14, the light one. And now the question is as the DNA molecules replicate, how will the heavy nitrogen from those parent molecules be distributed amongst the daughter molecules that are produced in successive duplications?

Stahl: Then some sensitive method for separating DNA, according to its density would be devised.

Meselson: I ran across an article about the centrifugation of cesium chloride solution to measure the molecular weight.

Stahl: If the DNA was in there with the cesium, it would find its position in the density gradient. If it was heavy DNA, it would tend to be down near the bottom of the tube where the cesium was concentrated and the density was high. If the DNA was light DNA, made of light isotopes, it would be higher up in the tube. You could think about it this way. If you jumped into the Great Salt Lake, as we all know you float, you go right to the top because you are less dense than the water. But if you have a bathing suit with pockets in it, and you stuffed some lead weights in your pockets, you’ll sink down. Cause you’re more dense than the water.

Now imagine that the salt in the Great Salt Lake is not uniformly distributed, but is concentrated near the bottom and rather less concentrated near the top. Now, if you put just the right number of heavy weights in your pocket, you won’t float because you’ll be too dense. You won’t float at the top and you won’t go all the way to the bottom because you’re not dense enough. You’ll instead come to rest somewhere, halfway between the top and the bottom, you will have found your place in that gradient. And that’s the very basis by which the experiment finally worked and worked so beautifully.

Meselson: And then it was just a question of looking in the centrifuge while it’s running. And when it reaches equilibrium to see where the heavy and light DNA are.

Stahl: All the makings were there, then to do the experiment itself, it was obvious that the experiment was going to give an answer.

Meselson: Driving it all was the fact that Frank wanted to know how life works.

Stahl: Yeah, yeah. I don’t know that drove it all but-

Meselson: Each person is trying to come up with something as a gift to the other guy.

Stahl: That’s true.

Meselson: I think.

Meselson: So it becomes a very connected relationship because the next day you want to have something to offer.

Stahl: Matt was ready to step out into an area, pretty heavily uncharted, to answer an important question. And the pieces had to be built as he went along.

Meselson: The prediction of the Watson and Crick model, was the two parent chains come apart. Each one makes a new daughter molecule and that’s replication. So that would predict that after exactly one generation, when everything has doubled in the bacterial culture, that you’d find the DNA molecules all have one old strand, which is labeled heavy. And one new strand, which is labeled light and therefore their density should be halfway between fully heavy and fully light, that would be the prediction for what you see at exactly one generation.

Stahl: What do you predict to see for the next generation? Well, each molecule would, again, separate its chains. One of which is heavy. The other of which is light and the only growth medium available is light growth medium. Then the light chain would make another light chain to go with it, a complement. The heavy chain would make another, a light chain to go with it. So after two generations you have DNA, half of which is half heavy. And the other half of which is all light. And fantastically, that’s exactly the result that one could see.

In order to say that the Watson-Crick model fits the data very well, but the other two models do not, we have to see what they’d predict. Start with the Dispersive Model. After one generation, the two molecules resulting would indeed be half heavy, but in the next generation, there would be a subsequent dispersion of the label. So you’d be getting molecules that were three quarters light, and one quarter heavy. And in each generation, the molecules would get lighter and lighter.

The fully Conservative Model simply imagined that duplex DNA fully heavy now, somehow created the appearance of a fully light duplex molecule in which both chains are made of light DNA. Most of the times when you get an experimental result, it doesn’t speak to you with such clarity. These pictures of the DNA bands interpreted themselves.

Meselson: It felt like a…supernatural. It felt like you were in touch with the gods or something like that.

Stahl: I remember I presented this result that summer early in the summer in France at a phage meeting, complete with the photographs of the density gradient bandings. And at the end of it, I stopped and there was total silence and somebody said, “Well, that’s it.”

Meselson: The intellectual freedom at Caltech. We could do whatever we wanted. It was very unusual for such young guys to do such an important experiment. So suddenly, whereas before that, like Max would be talking with Sinsheimer about the genetic code. And before we did our experiment, I was definitely not – at least I felt I wasn’t – supposed to be at those discussions. But afterwards, I could be a full member.

We had this wonderful house, big house across the street from the lab. And our roommates, we all, we talked about these experiments at almost every dinner. So we had this wonderful intellectual atmosphere, John Drake, Howard Temin. Why are you frowning?

Stahl: He told the dirtiest jokes I’ve ever heard.

Meselson: No that was Roger Milkman.

Stahl: Well you’re right, they held positions one and two.

Meselson: That’s true, that’s true, that’s true. So it was a very lively, intense, friendly atmosphere.

Stahl: It was lively enough and conveniently located enough that over time we had visits from William O. Douglas,

Meselson: Judge Douglas.

Stahl: Judge Douglas of the Supreme Court.

Meselson: And here Dick Feynman, probably one of the world’s greatest physicists at that time, or maybe ever, palled around with us. He came over to our big house and played his drums, sat down on the floor, played the drums. I’m just a graduate student and he’s the world’s greatest physicist, but that’s what it was like. It was a very friendly wide open place. Frank and I are very lucky.

The way I think of it is that there’s a river, which is a period of time when the fundamental things, the structure of DNA, how replication happens, the genetic code. And then, when these problems are solved. There are lots of little rivulets. The river divides into thousands of branches using these fundamental insights into how life works and applying them to specific questions, questions of disease, etc. So to me, with some exceptions, this was a really interesting time when it was still a big river.

Stahl: Also, now you can cut this out, but also the Meselsons, Matt’s parents, were kind enough to keep the liquor cabinet fully stocked at all times.

Stahl: My throat is a little bit?

Meselson: I have a cough drop.

Stahl: I don’t want a cough drop. I want a non-alcoholic beer. No, no, no.

Meselson: I require a margarita. I’ve worked for the CIA. I vaporized many people, including many of your friends, Big black beard, and blew out some of his pipe smoke and still holding his pipe stem in his teeth said, “Oh Matt, history is just what people think it was.”

Additional Resources

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

See the Explorer’s Guide to Biology for a first-person and in-depth description of Meselson and Stahl’s studies, as well as educational resources associated with their foundational key experiment. 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

Matt Meselson, Ph.D.

Professor of the Natural Sciences, Harvard University

Frank Stahl, Ph.D.

Emeritus Professor of Biology, University of Oregon

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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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The Meselson - Stahl experiment: Proof of Semi-Conservative Replication

Meselson & Stahl first grew bacteria for several generations in a medium containing only 15 N (" heavy " nitrogen). When examined in an analytical centrifuge, DNA isolated from these bacteria produced a single "heavy" band. Meselson & Stahl then transferred a portion of the culture to a new medium that contained only 14 N (" light " nitrogen). When DNA was isolated from these bacteria after one generation, they observed a single band that was "lighter" than the one obtained before; the "heavy" band was not observed in these bacteria. When DNA was isolated from the same culture after two generations, they observed two distinct bands of equal intensity, one with the same weight as seen in the previous experiment, and a new one still "lighter." When DNA was isolated from the same culture after three generations, this lightest band became the predominant one, and the middle band faded.

Meselson & Stahl reasoned that these experiments showed that DNA replication was semi-conservative : the DNA strands separate and each makes a copy of itself, so that each daughter molecule comprises one "old" and one "new" strand. Bacteria grown in "heavy" Nitrogen have been labeled on both strands entirely with "heavy" Nitrogen. After one generation in "light" Nitrogen, all of the DNA molecules comprise one "old heavy" and one "new light" strand, and have the same "heavy / light" molecular weight, which is less than that of "heavy / heavy" molecules. After two generations in "light" medium, the "heavy" and "light" strands separate, and both replicate with "light" nitrogen. Half therefore become "light / light", and half become "heavy / light" as in the previous experiment. In each successive generation, the proportion of “heavy” strands is reduced by half, and the “heavy / light” band gradually fades.

Homework : 1) Do you expect the lightest band strand to become still lighter with further generations of replication? Explain. 2) Suppose DNA replication were “ conservative ”: the parent strands separate, each makes a copy of itself, and the two new daughter strands come together as a new molecule and the old parent strands rejoin. Under those conditions, predict & draw the results of the Meselson – Stahl experiment.

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.

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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Front Matter
Table of Contents
Acknowledgments
Introduction
The Replication Problem
Meselson and Stahl
Twists and Turns
Crossing Fields:: Chemical Bonds to Biological Mutants
Dense Solutions
The Big Machine
Working at High Speed
The Unseen Band
One Discovery, Three Stories
An Extremely Beautiful Experiment
Centrifugal Forces
The Subunits of Semiconservative Replication
Images of an Experiment
Abbreviations Used in Notes

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Meselson–Stahl Experiment

Discovery and history, the background of replication of dna, semi-conservative replication , the dispersive hypothesis, the conservative hypothesis, the semi conservative, procedure and protocol followed by meselson and stahl, results and observation , ruling out conservative hypothesis, ruling out dispersive hypothesis, importance and application of meselson and stahl experiment, what did meselson-stahl experiment show, why were 14 n and 15n used in meselson and stahl’s experiment, how did meselson and stahl turn e.coli dna into heavy dna, what is the semiconservative model of dna replication.

The Meselson-Stahl experiment was a groundbreaking scientific study conducted in 1958 by Matthew Meselson and Franklin Stahl. This experiment provided strong evidence supporting the theory of semi-conservative DNA replication, which was proposed by James Watson and Francis Crick.

Read more about an Introduction to DNA Replication

After the brilliant work of describing the DNA structure, Watson and Crick also proposed a hypothesis that the DNA replication process is semi-conservative. This hypothesis was strengthened by the experiment of Meselson and Stahl in which they elucidated the nature of replication of DNA.

The cell was first discovered to be dividing by Hugo Von Mohl in 1835. Later, cell division was filmed and captured by Kurt Michel in 1943, who was renowned for micro-cinematography. It became evident that when the cell divides the nucleus also divides, and whatever material is present in the nucleus also undergoes division. So, the DNA present in the nucleus must undergo division. This division of DNA was named DNA replication. There were a lot of other hypotheses explaining the nature of DNA replication, but the Meselson-Stahl Experiment stood out the most as it supported the already existing hypothesis of Watson and Crick.

DNA is the basic code of life. When the cell divides it also divides and is transferred to the daughter cells. Just like the production of daughter cells, there are production daughter DNA strands. This production is simply the copying of the hereditary material and is called DNA replication. The process begins at specific sites where there is a characteristic nucleotide sequence.

Several enzymes have been found to assist in this process most important of which is DNA polymerase. This enzyme is of a single type in the case of eukaryotes while there are three types (DNA polymerase I, II, and III) in the case of prokaryotes.

The DNA polymerase I is relatively a short sized enzyme that plays only a supportive role, but DNA polymerase III is the main enzyme that causes replication in E. coli. DNA polymerase III has a large size, almost 10 times larger than DNA polymerase I. It moves along the DNA strand, adding nucleotides at a very quick rate of 1000 per second. However, DNA polymerase cannot itself initiate DNA replication. It requires the enzyme primase to create RNA primer, a complementary sequence of 10 nucleotides. DNA polymerase III identifies this and starts further adding nucleotides to complete the new daughter strand.

In this replication, it is cleared that when the DNA helix replicates it unwinds itself and turns out to create two strands. This unwinding is done by Helicase enzyme. One of the parent strands act as a template strand and on this, the complementary base pair attach to form a new stand. After the formation of this new strand or daughter strand, they wind together to form a supercoiled DNA double Helix with the help of topoisomerase enzymes. The same process happens with the other parent strand of DNA.

Thus, it was concluded that the new copies or replicas of the parent DNA helix consist of half (one of the strands) of the parent DNA. That is why it is called semi-conservative as it contains fifty percent of older or parent DNA.

Hypotheses Regarding DNA replication

Three hypotheses were tried to elucidate the DNA replication process up to their own extent.

The Dispersive hypothesis
The Semi-conservative Hypothesis

This hypothesis was based on the model, which was put forward by Max Delbruck, a German – American biophysicist. He introduced the concepts of physics at the molecular level in biology. According to the dispersive hypothesis, the parent DNA is digested into around ten or more segments. These segments are used as templates to make new segments. The old and new segments then mix and coil to form the daughter DNA strands.

Simply, this model suggested that the parent DNA strand would disintegrate, and the new strands formed will be the blend of the newly formed nucleotides (made by polymerase enzymes) and older nucleotides (from the parent DNA).

This hypothesis considers the DNA a template as a whole. The histone proteins bind to the DNA and revolve around it, producing a new DNA copy. In simple terms, the DNA duplex will remain the same. It will not be unwound and will create completely new copies.

As described earlier, it supports the hypothesis of Watson and Crick. Each of the parent DNA strands acts as a template and the new bases or nucleotides will form. This means it will be half new and half old i.e. semi-conservative replication.

In all of the above hypotheses, there is a prognosis about how the parent DNA would distribute itself. In essence, the conservative hypothesis tells us that the parent DNA will not mix up with the daughter DNA, but it will retain its identity. However, the daughter DNA might be new, but it will have the same sequence as the parent DNA.

The semi conservative hypothesis tells us that after the replication process the new DNA duplex formed will have fifty percent of the parent material and fifty percent new formed or daughter material (nucleotides), as complete single strands.

Whereas, the dispersive theory explains that the whole DNA duplex will divide itself in about 10 nucleotides in size and the old fragments will join randomly with the newly formed fragment or more precisely the nucleotides.

Experimental Process and Protocol

All three hypotheses were assessed by Mathew Meselson and Franklin Stahl. They performed experiments in California. As their experiment was highly based on the studies and hypotheses of Watson and Crick. They realized that DNA is made up of nucleotides. These nucleotides are further comprised of a phosphate group, deoxyribose sugar and most importantly nitrogenous base.

The nitrogenous bases are present in each nucleotide of DNA. All the bases present in those nucleotides have nitrogen. They realized it would be greatly helpful to tag the parent DNA.

The best way to tag it was to change the nitrogen atoms present in the parent DNA. They used a nitrogen isotope to make a difference between the daughter and parent strand. Due to this remarkable use of isotopes and biophysics, this experiment is regarded as the most beautiful experiment.

The experiment began with the preparation of a culture media for the bacteria. The microorganism chosen was E. coli . The culture media consisted of NH4CL and 15 N isotope of nitrogen. This isotope of nitrogen has a higher molecular weight than the normal existing nitrogen isotope 14 N.

When the E. coli was allowed to grow in the culture containing high molecular weight isotope of nitrogen, the bacteria started to incorporate 15 N atoms of nitrogen into its DNA. So, after several generations, the DNA of the bacteria grown in 15 N culture becomes denser than the normal bacteria grown in normal culture.

The other E. coli ware grown in a normal culture media containing the common isotope of nitrogen, the 14 N nitrogen. Thus, the DNA of this bacterial culture was less dense than 15 N culture media.

After completing enough generations, they transferred the bacteria from the 15 N media to 14 N media. They allowed the bacteria containing 15 N in their DNA to grow in a culture containing 14 N nitrogen.

They obtained DNA from the mixture and dissolved it in solution containing cesium chloride. Later, they centrifuged this mixture at high speed i.e. using the ultracentrifugation technique. The high centrifugal forces cause the ions of CsCl to produce a density gradient. The gradient is produced as the ions migrate to the bottom of the tube due to ultra-centrifugation. A constant density is established throughout the solution. The DNA samples start to float in the solution. After keeping the tube at rest for a while, the different DNAs of different densities maintain a specific position according to their densities.

As the DNA with 15 N isotope is heavier, it finds its place at the bottom of the tube. On the other hand, the DNA with 14 N isotopes of nitrogen finds its place on an elevated level in the tube. However, a type of DNA is also found in between the elevated level and bottom level.

Both the scientists concluded that if conservative hypothesis were to be true, then there would be DNAs of only two densities. As the parent DNA would have maintained its integrity and gave birth to a new daughter strand. However, they found three types of densities regarding DNA in their experiment.

According to the semi-conservative replication hypothesis, there would be a hybrid density DNA. As one strand will be of 14 N and one strand will be of 15 N isotope. However, concerning dispersive hypothesis the new DNA formed will be of intermediate density just like the semi-conservative. But, each of the strands from the double helix will not solely contain either 14 N or 15 N nitrogen, it will be a mixture of both isotopes.

To rectify these confusions, Meselson and Stahl sampled the second replication of 15 N bacteria in the 14 N medium. They followed the same procedure and protocol and put the DNA in the test tube with Cesium Chloride and established a density gradient.

It was found that there were DNAs of two different densities, unlike the first time when there were three. One of the two had intermediate density just like in the previous experiment and the second one had the density of pure 14 N isotope.

This result did not support the dispersive hypothesis of replication as it would have given one DNA having a density lower than the intermediate density found in the experiment.

This experiment provided modern biology with the knowledge of DNA replication. This knowledge granted the vision to peek into hereditary diseases and disorders. It is a simple yet provoking experiment that has been accepted by a lot of scientists.

Despite the affirmative result provided by the experiment of the Meselson-Stahl, it took a few years for acceptance by the scientific community. The Meselson and Stahl experiment did not only provide evidence for the semi-conservative theory which was put forward by Watson and Crick, this experiment also confirmed the Watson and Crick model of DNA structure. Thus, it strengthened the standpoint of Watson and Crick which was taking years to get accepted.

In 1952, a scientist studied the experiment by implementing it on the cancer researches. He did not negate the semi-conservative replication hypothesis. This further supported the experiment.

However, the only thing that fell short in this experiment was the proper explanation about the DNA subunits. This discovery of Meselson and Stahl allowed the scientists to discover the mode of transmission of DNA and follow up on the genetic disorders.

This experiment is simply based on tagging the DNA and separating them based on their densities relative to the solution created by using Cesium Chloride.

It was a known fact by then that DNA is made up of nucleotides and these nucleotides contained nitrogen.

Meselson and Stahl utilized the common nitrogen by replacing it with a heavier isotope so that they can identify the parent and daughter DNA in solution by mixing the DNAs of different densities.

They experimented using simple techniques as ultra-centrifugation and density grading.

This experiment is a great milestone in modern biology as those were early times when atomic physics was applied in biological studies. Meselson and Stahl made three major outcomes.

DNA is made up of two strands, which was based on the Watson and Crick Model
If the parent DNA has two strands, then each of the strand acts as a template and retains its integrity and forms a daughter strand. Thus, the semi-conservation is evident
Each of the parent DNA strand is shared by two daughter DNA.

In any case, the investigation helped researchers to clarify legacy by indicating how DNA saves hereditary data through all the progressive DNA replication cycles as a cell grows, divides and repeats the cycle.

Frequently Asked Questions

Meselson-Stahl’s experiment confirmed that DNA replicates semi-conservatively and ruled out the other proposed mechanisms of DNA replication.

Nitrogen is one of the abundant elements in DNA structure. Meselson and Stahl used the isotopes of N14 and N15 in the experiment to incorporate them into the DNA of newly growing organisms and separate DNAs of different densities to observe the mode of DNA replication.

Meselson and Stahl cultured E.coli in 15NH4Cl containing medium over many generations. As a result, 15N was incorporated into the bacterial DNA, converting it into heavy DNA.

According to this model, both strands of DNA segregate in the replication process, and each strand acts as a template to synthesize a new daughter strand. Resultingly, daughter DNA contains one parent strand and one new strand.

John Cairns to Horace F Judson, in The Eighth Day of Creation: Makers of the Revolution in Biology (1979). Touchstone Books, ISBN 0-671-22540-5 . 2nd edition: Cold Spring Harbor Laboratory Press, 1996 paperback: ISBN 0-87969-478-5 .
Watson JD, Crick FH (1953). “The structure of DNA”. Cold Spring Harb. Symp. Quant. Biol. 18 : 123–31. doi : 10.1101/SQB.1953.018.01.020 . PMID 13168976 .
Bloch DP (December 1955). “A Possible Mechanism for the Replication of the Helical Structure of Desoxyribonucleic Acid” . Proc. Natl. Acad. Sci. U.S.A. 41 (12): 1058–64. doi : 10.1073/pnas.41.12.1058 . PMC 528197 . PMID 16589796 .
Delbrück M (September 1954). “On the Replication of Desoxyribonucleic Acid (DNA)” (PDF). Proc. Natl. Acad. Sci. U.S.A. 40 (9): 783–8. doi : 10.1073/pnas.40.9.783 . PMC 534166 . PMID 16589559 .



				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

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Meselson and Stahl Experiment

Meselson and stahl experiment: introduction.

Replication is the process of generating a duplicate of anything. Regarding the replication model of DNA, or how DNA replicates, numerous hypotheses have been put forth by various scientists. The semiconservative replication of DNA was proposed by Watson and Crick. This model states that the DNA's two strands split apart and for the synthesis of a new strand, each strand serves as a template. Based on complementary base pairing with the template, the new strand is created.

The Meselson and Stahl Experiment was carried out to demonstrate the semi-conservative character of DNA replication . In 1958, Franklin Stahl and Matthew Meselson conducted research on E. coli bacteria.

Meselson and Stahl used the E.coli bacterium as a model system in their well-known investigations into DNA replication. They started by raising E. coli in nutritional broth or a medium that contained the "heavy" isotope of nitrogen, i.e., 15 N (an isotope of an element contains different number of neutrons in its nucleus). The bacteria absorbed the nitrogen when cultivated on a medium containing heavy 15 N and utilised it to create new biological molecules, including DNA.

The DNA of the bacteria's nitrogenous bases was all marked with heavy 15 N after many generations o f growth i n the 15 N . The bacteria were then transferred to a medium containing a "light" 14 N isotope and left to continue growing for a number of generations. 14 N , the sole nitrogen that would have been available for DNA synthesis following the transition, would have had to make up all of the DNA.

Meselson and Stahl were able to gather small samples in each generation, extract the DNA, and purify it because they knew how frequently E. co li cells di vided. Then, using density gradient centrifugation, they calculated the DNA's density (and, indirectly , its 15 N and 14 N co ncentration).

By spinning molecules like DNA at high speeds while another molecule, such as caesium chloride, generates a density gradient from top to bottom of the spinning tube, this technique divides molecules like DNA into bands. Minute changes, like those betwe en 15 N and 14 N label led DNA, can be detected using density gradient centrifugation.

Results of the Experiment

Generation 0

After centrifugation, DNA recovered from cells at the beginning of the experiment ("generation 0," right before switching to 14 N medium) formed a single band. The DNA should have had only a heavy 15 N at that time. Therefore, this finding was reasonable.

Generation 1

When centrifuged, DNA that had undergone one generation (one cycle of DNA replication) also created a single band. The density of this band, which was higher and between the heav y 15 N and light 14 N D NA, was intermediate.

From the intermediate band, Meselson and Stahl learned that the DNA molecules created during the initial round of replication were a mix of light and heavy DNA. The dispersive and semi-conservative models, but not the conservative model, agreed with this result.

Two different bands in this generation would have been predicted by the conservative model (a band for the heavy original molecule and a band for the light, newly made molecule).

Generation 2

Two bands emerged after centrifuging second-generation DNA. One was higher (seemed to be designated merely wi th 14 N), while the second was in the same place as the intermediate band from the first generation. Meselson and Stahl were informed by this finding that the DNA was replicated semi-conservatively.

The pattern of two separate bands—one a t a hybrid mole cule's place and the other at a light molecule's position—is exactly what we would anticipate for semi-conservative replication. A "purely light" molecule cannot be produced in dispersive replication since every molecule should contain both old and new DNA fragments.

Generation 3 and 4

Each hybrid DNA molecule from the second generation should, according to the semi-conservative model, result in both a hybrid molecule and a light molecule in the third generation, but each light DNA molecule should only produce more light molecules.

Therefore, over the third and fourth generations, we would anticipate the light band to get stronger and the hybrid band to get fainter (as it would represent a smaller portion of the total DNA because it would represent a larger fraction).

Meselson and Stahl Experiment Diagram

In semi-conservative replication, each of the two parental DNA strands acts as a template for new DNA strands to be synthesised. However, after replication, each parental DNA strand base pairs with the complementary newly synthesised strand, and both double-stranded DNAs include one parental or "old" strand and one daughter or "new" strand.

The word semi-conservative refers to the fact that the parental helix is half preserved and each parental single strand stays intact.

Conclusion of Semi-Conservative Replication of DNA:

Meselson and Stahl experiment proved that DNA replicates semi-conservatively, which means that each of its strands acts as a template for the synthesis of a new, complementary strand.

Despite the fact that Meselson and Stahl conducted their research using the bacterium E. coli, we now understand that semi-conservative DNA replication is a universal process that all life forms on Earth share. The cells are replicating their DNA in a semi-conservative manner.

FAQs on Meselson and Stahl Experiment

1. Why does semi-conservative replication occur?

The process of semiconservative replication takes place in every individual cell so that the genetic make-up of the parent cells can be maintained. We only resemble one another on a species level and are genetically related to our parents because of this; otherwise, if the semi-conservative mode of replication had not been used, events would have been extremely random. In other words, we are only related to one another on a species level. Due to the semi-conservative nature of DNA replication, every species manages to maintain its genetic purity.

2. What is the biochemical nature of the transforming principle?

To find the transforming principle, bacteriologists did a number of experiments.

Alcohol precipitated the transforming principle. This demonstrated that it wasn't a carbohydrate.

Proteases were unable to eliminate the transforming principle. So, the protein was not the cause.

The lipases were unable to remove the transforming principle. This demonstrated that it wasn't a lipid.

Ribonuclease could not inactivate the transforming principle. Hence, RNA was not effective.

Deoxyribonuclease may be used to inactivate the transforming principle.

DNA was the transforming principle. As a result, DNA was the genetic material.

3. What are the conservative and semi-conservative methods of replication of DNA?

The characteristics of the conservative model, the semi-conservative model and the dispersive model are as follows.

A single molecule containing both the parental copy of DNA and another copy including both strands of newly synthesised DNA is produced as a result of DNA replication, according to the conservative model of DNA replication.

Two parental strands unwind in the semi-conservative model and each serves as a template for the synthesis of its complementary strand, resulting in the formation of two molecules of DNA, each with a parent strand and a daughter strand.

According to the dispersive model of DNA replication, the parental DNA strands are randomly inserted into both daughter DNA molecules to produce hybrid DNA strands.

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COMMENTS

Mode of DNA replication: Meselson-Stahl experiment
The experiment done by Meselson and Stahl demonstrated that DNA replicated semi-conservatively, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand. Although Meselson and Stahl did their experiments in the bacterium E. coli, we know today that semi-conservative DNA replication is a ...
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. ... Experimental procedure and results. Nitrogen is a major constituent of DNA. 14 N is by far the most abundant isotope of nitrogen, but DNA with the heavier ...
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 ...
Meselson and Stahl Experiment
Meselson and Stahl Experiment Steps. Meselson and Stahl performed a series of an experiment, which includes the following steps: Growth of E.coli: First, the E.coli were grown in the medium containing 15NH4Cl for several generations. NH 4 provides the nitrogen as well as a protein source for the growth of the E.coli.
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 ...
5.6: The Meselson
Figure 5.6.1 Meselson - Stahl experiment interpretation. As this interpretative figure indicates, their results show that DNA molecules are not degraded and reformed from free nucleotides between cell divisions, but instead, each original strand remains intact as it builds a complementary strand from the nucleotides available to it. This is ...
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 ...
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 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 ...
How DNA Replicates
Support for video production was received from the Lasker and Rita Allen Foundations. To learn more about our commitment to Diversity, Equity, and Inclusion visit the "Our Mission" page. Follow us on. This key experiment by Meselson and Stahl established that DNA replicates through a semi-conservative mechanism, as predicted by Watson and Crick.
The Most Beautiful Experiment
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 ...
PDF The Most Beautiful Experiment in Biology The Replication of DNA in
Met Matthew Meselson in 1954. *To find more information on the various awards, see the Historical Timelines of Meselson and Stahl In 1957 he, along with Matthew Meselson, developed the technique of density gradient centrifugation, which led to the realization that DNA replication is semi-conservative through their experiment with E.coli DNA.
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.
Semi-Conservative Replication
The Meselson - Stahl experiment: Proof of ... Meselson & Stahl reasoned that these experiments showed that DNA replication was semi-conservative: the DNA strands separate and each makes a copy of itself, so that each daughter molecule comprises one "old" and one "new" strand. Bacteria grown in "heavy" Nitrogen have been labeled on both strands ...
PDF MeselsonandStahl:TheartofDNA replication I
doom, Matt came in,'' Stahl says. Me-selson 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
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-Stahl Experiment
https://explorebiology.org/collections/genetics/how-dna-replicatesMatt Meselson describes his experiment with Frank Stahl on DNA replication
Meselson, Stahl, and the Replication of DNA: A History of "The Most
In 1957 two young scientists, Matthew Meselson and Frank Stahl, produced a landmark experiment confirming that DNA replicates as predicted by the double helix s...
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 ...
Meselson-Stahl Experiment
The Meselson-Stahl experiment was a groundbreaking scientific study conducted in 1958 by Matthew Meselson and Franklin Stahl. This experiment provided strong evidence supporting the theory of semi-conservative DNA replication, which was proposed by James Watson and Francis Crick. Read more about an Introduction to DNA Replication.
Meselson and Stahl Experiment
4. 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.
Meselson and Stahl Experiment
The Meselson and Stahl Experiment was carried out to demonstrate the semi-conservative character of DNA replication. In 1958, Franklin Stahl and Matthew Meselson conducted research on E. coli bacteria. Meselson and Stahl Experiment. Meselson and Stahl used the E.coli bacterium as a model system in their well-known investigations into DNA ...

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Mode of DNA replication: Meselson-Stahl experiment

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Mode of DNA replication

Meselson and Stahl cracked the puzzle

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

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Three Proposed Models for DNA Replication

Defining the Models

Making Predictions Based on the Models

Straight or Circular?

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Meselson and Stahl: The art of DNA replication

A Partnership Begins

Not as Simple as It Seems

The Legacy of Elegant Peaks

How DNA Replicates

What's the Big Deal?

Learning Overview —

Bio-Dictionary Terms Used

Terms and Concepts Explained

Introduction

What Events Preceded the Experiment?

Setting Up the Experiment

Doing the Key Experiment

What Happened Next?

Closing Thoughts

Guided Paper

Dig Deeper 1: Alternatives to Semi-Conservative replication

Dig Deeper 2: The idea for using density for separation

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

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About the Film

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Matt Meselson, Ph.D.

Frank Stahl, Ph.D.

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

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DNA Replication and Meselson And Stahl's Experiment

Meselson and Stahl Experiment

Observation

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What are the different modes of replication of DNA?

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

What is the result of DNA replication?

What happens if DNA replication goes wrong?

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Meselson–Stahl Experiment

Hypotheses Regarding DNA replication

Experimental Process and Protocol

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Meselson and Stahl Experiment

Results of the Experiment

Conclusion of Semi-Conservative Replication of DNA:

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