meselson and stahl experiment of dna replication

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• Dna Replication Experiment

DNA Replication and Meselson And Stahl's Experiment

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

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

Meselson and Stahl Experiment

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

Semi conservative DNA Replication through Meselson and Stahl’s Experiment

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

Observation

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

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

Also Read:  DNA Structure

DNA Replication

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

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

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

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

DNA Replication Fork

Also Read:  DNA Replication

Frequently Asked Questions

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

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

What are the different modes of replication of DNA?

The different modes of replication of DNA are:

• Semiconservative
• Conservative

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

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

What is the result of DNA replication?

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

What happens if DNA replication goes wrong?

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Meselson and Stahl Experiment

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

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

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

Content: Meselson and Stahl Experiment

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

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

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

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

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

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

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

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

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

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

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

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

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

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How DNA Replicates

Matthew Meselson Franklin W. Stahl

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

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

What's the Big Deal?

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

Learning Overview —

Big concepts.

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

Bio-Dictionary Terms Used

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

Terms and Concepts Explained

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

Introduction

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

What Events Preceded the Experiment?

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

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

Setting Up the Experiment

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

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

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

Doing the Key Experiment

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

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

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

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

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

What Happened Next?

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

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

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

Closing Thoughts

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

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

Guided Paper

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Matt and Frank

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

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

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

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

Frank and Matt

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

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

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

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

We clearly needed a new strategy.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3) The replicative act results in a molecular doubling.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dig Deeper 1: Alternatives to Semi-Conservative replication

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

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

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

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

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

Dig Deeper 2: The idea for using density for separation

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

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

Matt describes briefly how this technique evolved

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

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

References and Resources

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

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

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

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

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

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

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

• Illustrations
• Problem Sets
• Calculators

You are here

Meselson–stahl experiment.

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

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

This is called semiconservative replication .

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

Three hypothesized patterns were proposed:

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

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

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

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

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

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

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

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

Related Content

• Complementary Nucleotide Bases
• DNA Polymerase
• Semiconservative Replication
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• Book Review
• Published: 01 March 2002

Meselson, Stahl and the Replication of DNA: A History of “The Most Beautiful Experiment in Biology”

• Bruce Stillman 1  

Nature Medicine volume  8 ,  page 211 ( 2002 ) Cite this article

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Meselson, Stahl and the Replication of DNA

• Frederic Lawrence Holmes

Great experiments either prove a previous notion, or they reveal unexpected results that lead to new ideas. In science, ideas are propagated and the very best of them survive for many years, if not forever. Experiments are, by their very nature, often transitory and useful but for a moment in time. However, at least one notable experiment is an exception: the famous Meselson and Stahl experiment. In a recent labor of love, Frederic Lawrence Holmes delves into this experiment, telling us how it came about, how it was conceived, how it was executed and what it meant at the time. Along the way, one gets a glimpse of what it was like to do science in the earliest days of molecular biology and a sense of the social aspects of science in those heady times.

The second of the famous papers by Jim Watson and Francis Crick deals with the implications of the double-helix structure for inheritance and states that “each chain then acts as a template for the formation on to itself of a new companion chain so that eventually we shall have two pairs of chains, where we only had one before”. Therein, they proposed that the DNA unwound and each strand was a template for the synthesis of a complementary strand, begetting two identical helices. They suggested that DNA might replicate in a semi-conservative manner, rather than the alternative conservative mode whereby the parental double helix remained intact and the new double helix was identical to the parent, but composed of entirely new strands. The Meselson and Stahl experiment demonstrated that Watson and Crick were correct in their assumption.

It seems difficult these days to comprehend that there was ever any doubt about how DNA must replicate. But masterfully, and in great detail, Holmes takes us back to the discourse that emerged immediately after the double-helix revelation. Many were concerned about what the great Max Delbrück thought of the double helix, and although he enthusiastically spread the word about its structure, true to form, Max had a problem: the “untwiddling problem”. How could the two strands that were intertwined so many times separate during replication? He was not only concerned about the problem, as were Watson and Crick, but he proposed a complicated (and incorrect) solution in a Proceedings of the National Academy of Sciences paper in the Spring of 1954.

Holmes' well-written book describes every detail from thenceforth. The chance meeting of Matt Meselson and Frank Stahl at Woods Hole, the seminar by Monod that induced Meselson to think about density transfer, the trials of experimentation and of course the “beautiful experiment” itself. Although dense, the story is worth reading to understand what science was like in the 1950s and how a great experiment came about. It also describes the environment at Caltech during that era, scientifically exciting, but socially bleak. I assume the social environment in Pasadena has improved, but clearly the science there remains as strong as it was. Students who do science, or those who study the process will learn much from this book on how great science can be accomplished.

What struck me while reading this treatise was the remarkably open exchange of ideas between the early phage investigators, via letters and discussions at meetings. Scientists traveled (and reveled) more than I would have thought, a common thread that has emerged in other books I have read about the early phage days. For example, Holmes reports that Meselson and Stahl wrote many times to Jim Watson and others about the design and progress of their experiments. Obviously Watson had a more than passing interest in the matter, but more interestingly, Meselson and Stahl wrote to and visited Gunther Stent at Berkeley to discuss their progress. They did this even though Stent was working on the replication problem and favored the Delbrück proposal that DNA replication was not semi-conservative. We should learn from history, because unfortunately, in modern molecular biology where scientists are not as technique-limited as they once were, the free exchange of ideas is in danger of being lost.

The measure of a great technique is what it reveals and whether it lasts. The Meselson and Stahl experiment is still in wide use today. It has been used to demonstrate the distributive nature of histone deposition during chromosome replication and most recently to study the mechanism and timing of replication of the entire genome of the yeast Saccharomyces cerevisiae . Very few experimental methods have survived as long as the density-transfer idea. Thus, I expect that Holmes' book will be read for many years to come, and justifiably so.

Also reviewed by Sydney Brenner

Salk Institute for Biological Studies La Jolla, California, USA

In these days of high throughput science, when advances in technology have literally given us the power to make atom-by-atom descriptions of all living matter, it is refreshing to look back at an earlier time, when advances in science required both a good idea and the means to show it was true. We were like Houdinis, strapped in chairs with our hands tied behind our backs trying to escape from locked rooms. This book is the history of the Meselson–Stahl experiment—the most beautiful experiment in biology—and reconstructs both the background and the event itself in a most meticulous and admirable way. Although we learn about the revolution in biology consequent upon the discovery of the double helix, it is not history in the large but rather history on the minute scale of what actually happened in the creation and execution of the experiment. The author has had access both to the notebooks and the memories of the scientists as well as to others and he has marshaled all of this detail into a narrative that is interesting and informative.

When the double helical structure of DNA was proposed, the intertwining of the strands created an objection in the minds of some who became concerned that the strands would have to be unwound in order for them to be replicated. Max Delbruck, in particular, was most troubled by it. It was fortunate that, at the time, people did not know that there were DNA molecules that were closed circles, because they would have declared the replication model proposed by Watson and Crick impossible. Somewhere the book says that there was a small band of enthusiastic supporters who were not troubled by this difficulty. I was one of them and took the view that if it were a problem, biological systems would have found a way to solve it. Indeed, I think it was Leslie Orgel who said that nature would have invented an enzyme to do it, a most perceptive insight.

The consequences of the replication model were clear: after one replication step two molecules would be present, each with one old and one new strand. How could one prove this? I met Matt Meselson outside Blackford Hall in Cold Spring Harbor in September 1954 when he had already conceived of the idea of doing the experiment with heavy isotopes using some sort of density centrifugation to separate the molecules. Frank Stahl knew how to work with phages and the partnership was formed. However, Meselson had to complete his PhD thesis research in crystallography, and while making the transition from physical chemistry to biology, he kept detailed notes about what he was reading in a workbook. The evolution of his thinking can be followed from these books.

After spending time trying to do the transfer experiment with 5-bromouracil-labeled bacteriophage T2, density-gradient ultracentrifugation became possible and they switched to using bacteria and 15 N labeling. They were able to show that the difference in density between light 14 N- and heavy 15 N-labeled DNA was sufficient to allow a molecule of intermediate density to be resolved, whereupon Meselson decided to do a double-transfer experiment from heavy to light and light to heavy medium against the advice of Stahl who had to go to an interview in Missouri. Meselson also added several controls and labeled the tubes from this large series of experiments with a complicated code before proceeding to analyze them in the ultracentrifuge. His memory was that the experiment had worked, but an examination of the original films showed that his recollection of the result was wrong. None of the films showed the expected three bands that Meselson thought he saw when he rushed over to announce the result at a party being held at his house. Of course, later experiments gave the expected result.

It could be said that if historians have the benefit of hindsight, scientists have the advantage of foresight. Meselson had sketched the expected result before doing the experiments and I think he superposed in his mind the individual results of his experiments to generate an answer compatible with it. All experimentalists know you have to do an experiment four times. The first one is a complete mess and shows only a hint that it might have worked. The second one is better but still messy. Then you do it the third time for the book. This is when you forget to add a reagent, or mix up the tubes or the centrifuge leaks. That is why there is always a fourth time.

I urge every young scientist to read this book. In 1957, when the experiment was performed, Meselson was 27 and barely with a PhD in chemistry. Frank Stahl was 28 and a postdoctoral fellow at the California Institute of Technology. Both were doing an experiment that had nothing to do with their official programs of research. They simply went ahead and did it. They filled out no forms, made no applications, had no reviews. They only had the judgments of their real scientific peers.

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Density matters: The semiconservative replication of DNA

Philip c hanawalt.

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Perspective

Issue date 2004 Dec 28.

The semiconservative mode of DNA replication was originally documented through the classic density labeling experiments of Matthew Meselson and Franklin W. Stahl, as communicated to PNAS by Max Delbrück in May 1958. The ultimate value of their novel approach has extended far beyond the initial implications from that elegant study, through more than four decades of research on DNA replication, recombination, and repair. I provide here a short historical commentary and then an account of some developments in the field of DNA replication, which closely followed the Meselson–Stahl experiment. These developments include the application of density labeling to discover the repair replication of damaged DNA, a “nonconservative” mode of synthesis in which faulty sections of DNA are replaced.

DNA replication is arguably the most fundamental process required for the proliferation of all living cells. During cell division, each daughter cell must receive essentially the same genetic information that was encoded in the DNA of the parent cell. This conclusion means that DNA replication must generate a perfect copy of the genomic DNA complement. Convincing experimental evidence for a “semiconservative” mode of DNA replication was first provided by the elegant experiments of Matt Meselson and Frank Stahl ( 1 ), in which differential labeling with nitrogen-15 ( 15 N) and nitrogen-14 ( 14 N) was used to resolve parental and daughter DNA molecules by equilibrium sedimentation in a CsCl density gradient. By “semiconservative,” it is meant that the parental DNA subunits are conserved but that they become equally distributed into daughter molecules as replication proceeds. It was originally thought, and is now known to be true, that these “subunits” are the complementary single strands of the double-helical DNA duplex.

A comprehensive historical description of the collaboration between Meselson and Stahl, the milieu in which they worked, and their remarkable path to success was prepared by the late Frederic Lawrence Holmes and titled Meselson, Stahl, and the Replication of DNA : A History of “ the Most Beautiful Experiment in Biology ” ( 2 ). This account highlights the personalized side of the story and provides a wonderful example of how seminal research is actually done. The crisp rendition of experiments and their clear-cut interpretations in the published journal article cannot begin to reveal the tortuous path of the research, from the germination of ideas, through the disappointments and surprises as the experimental results appear, to the ultimate success of the project.

Speculation about how DNA might replicate directly followed the proposal by James Watson and Francis Crick for its double-helical structure, in which the pairing of bases through hydrogen bonds and stereochemistry ensured that the two strands would be complementary ( 3 ). A thymine in one strand is always paired with an adenine in the other, and correspondingly, cytosine is always paired with guanine. That part of the model incorporated Erwin Chargaff's “rules” ( 4 ), based on the relative frequencies of these bases in DNA. Reflecting on their duplex DNA model, Watson and Crick stated, “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” ( 3 ). Thus, the strands might separate and then serve as templates for the synthesis of the respective complementary strands—a semiconservative mode of replication, in which each daughter DNA molecule would consist of one “old” strand and one “new” one.

Whereas the suggested mechanism seemed plausible, it was not immediately apparent how it might be rigorously tested. Furthermore, there were some rather vexing topological problems with which to contend. The DNA strands in the Watson–Crick helix are wound about each other in a “plectonemic” manner—which means that “for any winding number greater than zero, the `braid' consisting of the two chains cannot be combed” as Max Delbrück and Gunther Stent ( 5 ) pointed out in their early review on the subject. The Watson–Crick scheme assumed that unwinding and replication must proceed pari passu , with all three arms of the duplex DNA rotating at a replication fork. Another model suggested that periodic double-strand breaks would permit short sections of the duplex DNA to spin and then rejoin with the respective strand terminals of the same polarity. Although we now appreciate that an unprotected double-strand break in DNA is a very serious threat to cell viability, it has turned out that transient strand breaks are indeed the means by which the topological problem is resolved. As is often the case, when we are unable to explain how a plausible biochemical model might work, it may be because we have yet to discover an essential enzyme, in this case, topoisomerase. Topoisomerases are DNA “nicking-closing” enzymes and the type II topoisomerases, such as gyrase, in particular, are designed to pump negative supertwists into the DNA ahead of an advancing replication fork, thus relieving the unwinding stress and facilitating processive separation of the two strands ( 6 ). Otherwise, positive supertwists would accumulate ahead of the replication fork during replication as the parental strands are separated behind it. The topological problems of unwinding parental DNA strands and segregation of daughter DNA duplexes were resolved many years after the basic mechanism of DNA replication was revealed ( 7 ). Provocative, and perhaps clairvoyant, was the statement of Delbrück and Stent ( 5 ) regarding the putative semiconservative mode of DNA replication—“if it were possible to label differently the new material synthesized in each generation, then one could read off in each duplex the ages of the two chains.”

In an exemplary set of experiments (of which Max Delbrück was surely aware) in late 1956, J. Herbert Taylor et al. ( 8 ) labeled the chromosomes of Vicia faba (English broad bean) with 3 H-thymidine, and then followed the distribution of the tritium label through successive generations of duplication in nonradioactive medium, by using autoradiography. The remarkable conclusions from this study were “that the thymidine built into the DNA of a chromosome is part of a physical entity that remains intact during succeeding replications...” and “that a chromosome is composed of two such entities probably complementary to each other,” and “that after replication of each to form a chromosome with four entities, the chromosome divided so that each chromatid (daughter chromosome) regularly receives an `original' and a `new unit.”' Taylor appreciated, of course, that the “chromosome is several orders of magnitude larger than the proposed double-helix of DNA.” Nevertheless, he had demonstrated that eukaryotic chromosomes divide semiconservatively, in accordance with the predictions of the Watson–Crick model, if a chromosome contained a duplex DNA molecule.

The Germination of an Idea: Meselson Meets Stahl

Matt Meselson began his graduate study in chemistry at the California Institute of Technology (Caltech, Pasadena, CA) in 1953. He joined the laboratory of Linus Pauling and ultimately completed part II of his Ph.D. thesis ( 9 ) on The Crystal Structure of N, N′ dimethyl malonamide , to determine whether the peptide groups contained in this molecule were planar, and thus in accord with Pauling's resonance theory. That chapter of his thesis is less widely known than is part I, which was titled Equilibrium Sedimentation of Macromolecules in Density Gradients with Application to the Study of DNA. As a student in Pauling's course on the chemical bond, Matt became interested in the comparative strength of hydrogen bonds when the natural hydrogen was replaced with the heavy isotope, deuterium. While developing his interest in how living organisms might fare if they incorporated deuterium into their molecules, Meselson happened to attend a seminar at Caltech by Jacques Monod, who raised the question of whether the then-poorly understood phenomenon of induced-enzyme synthesis really involved new protein synthesis. Meselson speculated that if bacteria could be grown up in deuterium (heavy) water, and then transferred to ordinary water at the same instant that an “inducer” was added, any new proteins should be of “normal” density and the density difference between “old” and “new” proteins might permit their separation. In fact, if one centrifuged the proteins in a solution of intermediate density, then the “old” protein might sink whereas the newly synthesized protein should float. Later that year, he turned his attention to the DNA replication problem, after a lively discussion with Max Delbrück about the Watson–Crick double helix and possible modes for its duplication. It occurred to Matt that the same approach he had envisioned for protein synthesis might also be applied to study DNA replication. He decided at that point that he wanted to devote his energies to determine whether DNA, indeed, replicated in the manner predicted by Watson and Crick. Unrelated to that goal, he spent the summer of 1954 at the Marine Biological Laboratory at Woods Hole, MA, assisting Jim Watson with some titration experiments that were designed to provide possible support for a double-helical structure of RNA, analogous to the structure of DNA. Frank Stahl, then a graduate student in biology from the University of Rochester (Rochester, NY), was also at Woods Hole for the summer to take a physiology course. They met while Frank was sitting under a tree working on a problem in bacteriophage genetics. Fig. 1 is a photo of Matt and Frank 42 years later, standing at the same place. Whereas Matt was still quite naïve about bacteriophage genetics, he possessed the skills in calculus to help Frank solve the problem. As they became acquainted, Matt then raised the possibility that they might work in collaboration on the DNA replication problem—using phage DNA, to take advantage of Stahl's expertise. He also suggested using deuterium as a heavy label in a one-step growth experiment (by analogy with his earlier thoughts about density-labeling proteins), and to then centrifuge the sample in a solution of an appropriate density to separate the “light” DNA at the top of the tube from the “heavy” DNA at the bottom. However, they both soon recognized the complicating problem of doing their replication experiment with phage, because of the extensive recombination known to occur, which might be expected to reshuffle parental and daughter DNA, and thereby confuse the analysis. Fortunately, Stahl was already planning to go to Caltech for his postdoc, so they would be able to continue their discussion and work together there, perhaps to develop their strategy by using a “simple” cell system, the bacterium Escherichia coli .

Photograph taken by F. L. Holmes of Matt Meselson and Frank Stahl in 1996, standing at the site where they met at Woods Hole 42 years earlier (figure 14.1 in ref. 2 ). Courtesy of the Holmes family.

Matt wanted to find out more about the chemical nature of the monomer precursors for DNA, and in the course of that literature search he learned about 5-bromouracil (5BU), an analog of thymine, which bacteria could incorporate during DNA synthesis in place of thymine. 5BU is equivalent to thymine except that bromine is substituted for the methyl group at the C5 position: the bromine conveniently has nearly the same van der Waals radius as a methyl group. Because of the different degree of ionization between 5BU and thymine, Matt considered that he might be able to separate 5BU-labeled molecules from those containing thymine by electrophoresis. However, more importantly, he appreciated the fact that 5BU would make the DNA containing it significantly heavier than normal thymine-containing DNA. He then considered using 5BU as a density label for DNA to follow its replication by the scheme considered earlier.

Matt became acquainted with Jerry Vinograd, who was the ultracentrifugation “guru” at Caltech, and he learned to operate the state-of-the-art Beckman Spinco Model E analytical ultracentrifuge ( Fig. 2 ). With Vinograd's initial tutelage, Matt tried sedimentation of DNA in a 7-molal solution of the heavy salt, CsCl—his idea was still that an experiment could be performed with a density label and that “light” DNA should float and that the density-labeled heavy DNA would sink in a solvent of the appropriate density. However, they were both amazed at how rapidly a salt gradient formed during the high-speed centrifugation and, furthermore, that the DNA migrated to a narrow band within the gradient. The band formed at the position of the buoyant density of the DNA in that stable salt gradient.

Photographs of Jerome Vinograd and Matt Meselson. ( a ) Jerome Vinograd by “his” Spinco Model E analytical ultracentrifuge, serial no. 186. (Courtesy of the Caltech Archives.) ( b ) Matt Meselson at the controls for the UV optics and photography system of Model E no. 186 used for the classic experiment. (Courtesy of the Caltech Archives.)

The concept of equilibrium sedimentation in density gradients generated during the approach to equilibrium of a low molecular weight solute (e.g., CsCl) was elaborated by Meselson et al. ( 10 ) in a paper communicated to PNAS by Linus Pauling in May 1957. The figures in that paper and the theoretical calculations are essentially part I of Meselson's Ph.D. thesis, which, interestingly, provides no preview of the intent to apply density labeling to the study of DNA replication. The paper focuses instead on the nature of the band structure and the fact that the concentration distribution of a single macromolecular species in a constant density gradient should be Gaussian, and that the standard deviation of that band is then inversely proportional to the square root of the macromolecular weight. The model was remarkably correct, as tested with homogeneous DNA of known molecular weight from bacteriophage T4. This paper also documents the first analysis of the density distribution of DNA containing 5BU, obtained from T4-infected cultures of E. coli grown in media with this thymine analog. The 5BU fully substituted DNA molecules banded at a density of 1.8 g/cm 2 , whereas those of normal thymine-containing T4 bacteriophage DNA were well separated from these at 1.7 g/cm 2 . Although there was no mention of using this approach to study DNA replication, the application to study intact viruses and smaller molecules like proteins is discussed in this pioneering report on density gradient sedimentation.

The Classic Experiment

Matt and Frank were well on their way to design their landmark experiment on DNA replication. They might have used 5BU as the density label but they became concerned about the deleterious effects of its mutagenicity and cellular toxicity, as well as problems in obtaining uniform labeling, so they decided instead to use a synthetic growth medium in which the sole source of nitrogen was 15 NH 4 Cl.

The bacterium E. coli was grown for many generations in 15 NH 4 Cl medium so that the DNA would be essentially fully labeled with the heavy isotope 15 N. Then, the medium was diluted with a 10-fold excess of 14 NH 4 Cl as exponential growth continued. Samples were taken from the growing bacterial culture at various times to analyze the distribution of DNA densities in a CsCl gradient. There was initially a single band at the 15 N heavy DNA position, and then a second band began to appear at a position half way between the density of 15 N DNA and that of 14 N DNA. The parental 15 N band disappeared with time as this “hybrid” band formed. At precisely one generation (or division cycle), only the intermediate density hybrid band was present. It was then important, indeed essential, that the experiment was continued for a second generation, thereby to establish that when the hybrid DNA replicated in the 14 N medium, equal amounts of “light” and hybrid DNA were present at the completion of that second cycle. Thus, the hybrid DNA was continuously regenerated during replication and the amount of light DNA increased with each round of replication. There was the profound implication that the constant amount of hybrid molecules will be maintained “forever” as successive cell divisions continue.

At this point it was clear that “the nitrogen of a DNA molecule is divided equally between two subunits which remain intact through many generations,”... “the subunits are conserved,” and that each daughter molecule receives one parental subunit, according to the scheme shown in Fig. 3 [which is figure 5 in the Meselson–Stahl paper ( 1 )]. An essential requirement of the model is that the two subunits must separate. Meselson and Stahl ( 1 ) provided convincing evidence of this through thermal denaturation studies in which the DNA samples were kept at 100°C for 30 min in the CsCl before centrifugation. The hybrid DNA clearly resolved into two bands at the respective positions of heat-denatured 15 N-DNA and 14 N-DNA under these conditions. Furthermore, the broader Gaussian bands of the denatured DNA indicated a reduction of roughly one-half in the molecular weight from that of duplex DNA, as consistent with the view that the subunits are single DNA strands. Nevertheless, the conclusions were stated with cautious restraint, leaving open the questions about the nature of the molecular structures of the subunits and the relationship of these subunits to each other in a DNA molecule ( 1 ).

Interpretation of what the density labeling data actually confirm in terms of a model for DNA replication (figure 5, from p. 677 of ref. 1 ). [Reproduced with permission from ref. 1 (Copyright 1958, PNAS).]

I first learned of the Meselson–Stahl experiment while I was a graduate student at Yale, when I attended the second annual meeting of the Biophysical Society, in Cambridge, MA, in early 1958. In a contributed-paper session, Matt was accorded two successive 15-min slots for his talk, as I recall, as the Chair announced that this next presentation was going to be of very special significance. It was indeed an exciting and generally convincing presentation: clearly the highlight of the meeting for me and for most others.

Caveats About the Proof

In Meselson's talk and in their PNAS paper, as noted above, Meselson and Stahl ( 1 ) were very careful about what they could actually claim from their experiment. Figure 5 in their paper implies no more and no less about what can be concluded, even though the most nonobvious and straightforward assumption is that the conserved “subunits” must be single DNA strands. Was the Meselson–Stahl experiment definitive proof for semiconservative replication of DNA? In principle, the answer is yes, but there were additional important controls to be carried out. Although it was by no means a favored interpretation of the results, it was technically possible that the conserved “subunits” of DNA constituted an end-to-end association of parental DNA with newly synthesized daughter duplex DNA “subunits,” rather than lateral association of parent and daughter DNA strands. This unlikely scenario was ruled out by Meselson's graduate student, Ron Rolfe, who showed that sonication to intentionally break the linear hybrid DNA into shorter lengths, did not alter the density of the DNA ( 11 ). Another unlikely scenario was promoted by Liebe Cavalieri et al. ( 12 ), who argued that the conserved “subunits” might be double-stranded DNA and that the hybrid DNA would then consist of the lateral association of two duplex DNA helices to form a four-stranded structure. Following up on several years of heated debate, definitive exclusion of the Cavalieri model was ultimately provided from the work of Robert Baldwin and Eric Shooter ( 13 ), who studied the melting of hybrid DNA, in which one subunit was labeled with 5BU. The melting profile was that expected for DNA in which the subunits were single strands rather than double helices. Meselson's graduate student at Harvard, John Menninger ( 14 ), had shown by low-angle x-ray scattering that the linear density of E. coli DNA corresponded to two chains rather than four.

Essential to the success of the Meselson–Stahl experiment was the fragility of the rigid linear DNA molecule, and the effect of extensive shearing of the DNA when it was handled; particularly as the sample was injected into the ultracentrifuge cell through a hypodermic syringe, now known to impose high shear. The molecular mass of the DNA fragments studied was only ≈7 × 10 6 Da. If the entire bacterial chromosome could have been isolated intact in these gradients, the interpretation of the results might have been more complicated, because there would have been a gradual shift with time of DNA from the parental to hybrid density, as sequential replication proceeded around the circular genome.

A few years after the classic replication paper was published, Meselson and Jon Weigle ( 15 ) used the combination of 15 N and 13 C to prepare heavily density-labeled λ phage DNA to determine whether recombination (with normal density λ phage) involved a “copy choice” mode or one of “breakage and reunion.” In other words, was there any parental DNA in recombinant phage? The answer was that both chromosomal subunits are broken during recombination and that recombination occurs by chromosome breakage (although other mechanisms were not excluded). These studies used preparative CsCl density gradient ultracentrifugation and the enhanced resolution afforded by using two density labels. However, the procedure to prepare the 13 C-labeled precursors was extremely tedious, until 13 C-labeled glucose became commercially available some years later. Stahl and colleagues ( 16 ) then used 15 N 13 C double labeling in a series of important studies to elucidate relationships between the processes of recombination and replication in λ phage. Their initial paper in this series ruled out the so-called master-strand model for replication, another unlikely alternative to the Watson–Crick scheme. It eventually became apparent that 5BU is a very convenient choice for density labeling DNA—for many reasons, including the fact that 5BU (fully replacing thymine) achieves a density shift roughly equivalent to the combined use of 15 N, 13 C, and deuterium, and at lower cost.

During my postdoc with Ole Maaløe in Copenhagen, in 1959, we found that if protein synthesis was inhibited in growing E. coli , then only a limited amount of DNA synthesis could occur, and we postulated that this constituted completion of those cycles of replication underway, without initiation of any new ones. The definitive proof of that hypothesis came from density labeling studies with 5BU—in which we showed that in the absence of protein synthesis only hybrid density DNA appeared during replication—thus, no second round could have been initiated to yield DNA molecules with 5BU in both strands ( 17 ). My studies with 5BU labeling prompted additional speculation about the detailed mode of DNA replication—why did one not observe DNA molecules in which replication forks had been caught midway? These molecules would be predicted to appear in the density gradient somewhere between the parental DNA density and that of the hybrid band.

Meeting Meselson

When I arrived at Caltech in September 1960 for my second postdoc (with Robert Sinsheimer), I immediately sought out Matt Meselson—and fortunately caught him for several short discussions before he departed in early 1961 for his faculty position at Harvard. We discussed the nature of the E. coli chromosome and Matt speculated that it might consist of short segments of DNA held together by some sort of protein “linkers” that could help with the topological unwinding problem. John Cairns ( 18 ) used tritium autoradiography several years later to provide evidence that the bacterial chromosome consisted of one intact closed circular molecule of DNA, and that DNA replication proceeded around the circle from one (or at most two) growing points. The conclusion that the chromosome consisted of double-stranded DNA was based on the contour length of the circle, compared with the cellular DNA content. The possibility of “linkers” between DNA segments could not be excluded, however, because of the low resolution of the technique.

I thought that a possible explanation for the lack of “intermediate” density DNA between parental and hybrid bands in the Meselson–Stahl experiment could be that the replication of a DNA “segment” was essentially “all or none”—it happened so rapidly that only a negligible fraction of the DNA segments might be caught in the act. However, my student, Dan Ray, and I ( 19 ) were able to isolate partially replicated DNA fragments from growing E. coli , by using 32 P pulse labeling along with 5BU incorporation, and a very gentle cell lysis procedure before preparative CsCl equilibrium sedimentation. After mild shearing of those fragments, the labeled DNA was resolved into hybrid and parental density bands, suggesting that the replication fork DNA might be unusually sensitive to breakage. The intermediate density 32 P pulse-labeled DNA fragments could also be chased into the hybrid band when excess 31 P was added to the growing cells ( 19 ). I then reasoned that if we could stall replication forks at obstructions in the template, we might stabilize and recover those partially replicated molecules for further analysis. My student, David Pettijohn, and I ( 20 ) examined the density distribution of DNA during labeling with radioactive 5BU in the period immediately after UV irradiation of the bacteria, to introduce cyclobutane pyrimidine dimers known to arrest DNA synthesis. We did indeed find a substantial amount of intermediate-density DNA but, curiously, there was also a significant amount of nascent DNA label at the parental density. Rebanding the parental density DNA in a second CsCl gradient verified the presence of 5BU-containing DNA with little or no evident density shift. The plausible explanation became apparent when I discussed our experiments with my former graduate mentor, Richard Setlow ( 21 ), who had just discovered that cyclobutane pyrimidine dimers are released from the chromosomal DNA in UV-resistant bacteria: he postulated an excision-repair scheme for damaged DNA. We were evidently observing the patching step in this putative process of excision repair, and the lack of a density shift was because of the fact that the patches synthesized by repair replication were too short to appreciably shift the density of the DNA fragments containing them. ( Fig. 4 ) Thus, the approach developed by Meselson and Stahl ( 1 ) to demonstrate semiconservative DNA replication was used to first document the “nonconservative” repair replication of damaged DNA ( 20 ). Intentional shearing of the “repaired” DNA by sonication did result in a measurable density shift, which, when combined with molecular weight determinations, could be used to estimate the patch size.

Distinguishing semiconservative replication from nonconservative repair replication by using density labeling with 5BU.

As with the excision repair of damage (like cyclobutane pyrimidine dimers), the heteroduplex regions generated during genetic recombination were thought to provoke localized excision of a tract of nucleotides from one strand followed by repair synthesis to fill the gap. The excision repair of mismatched bases was also postulated, and Wagner and Meselson ( 22 ) obtained genetic evidence that, although well separated mismatches were repaired independently, sometimes those separated by <2,000 nt could be repaired by a single event, if these were on the same DNA strand.

The approach pioneered by Meselson and Stahl ( 1 ) continues to be widely used for research in the fields of DNA replication, recombination, and repair. It is the method of choice when one wishes to physically separate the newly synthesized DNA from DNA existing before an appropriate density label is introduced into a culture of growing cells or a replication system in vitro . It has become a classic approach for the biochemical detection of DNA strand exchange in recombination, although it does not approach the sensitivity of genetic analysis. Also, it is still used for the quantification of nucleotide excision repair in a variety of prokaryotic and eukaryotic cell systems. In a 1959 letter to Frank Stahl, Matt wrote that “CsCl has an inexhaustible number of golden eggs to lay.” That statement indeed has proved to be true.

This Perspective is published as part of a series highlighting landmark papers published in PNAS. Read more about this classic PNAS article online at www.pnas.org/misc/classics.shtml .

Abbreviation: 5BU, 5-bromouracil.

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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.

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The Most Beautiful Experiment: Meselson and Stahl

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

• General Public
• Educators of H. School / Intro Undergrad

Talk Overview

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

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

Speaker Bio

Frank stahl.

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

Matthew Meselson

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

More Talks in Genetics and Gene Regulation

Related Resources

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

Teaching resources from XBio: How DNA Replicates

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

Reader Interactions

ANGELA DIXON says

February 8, 2021 at 9:39 pm

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

Marieke Mackintosh says

March 23, 2021 at 12:08 pm

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

Neeraja Sankaran says

August 30, 2022 at 7:00 am

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

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1958: Semiconservative Replication of DNA

Matthew Meselson and Franklin Stahl demonstrated that DNA replicates semiconservatively, with each strand in a DNA molecule from the parent generation pairing with a new strand in the daughter generation. Each "parent" strand of DNA served as a template for the synthesis of a new strand of DNA.

More Information

Meselson, M., Stahl, F.W. The Replication of DNA in E. coli. Proc Nat Aca Sci USA, 44: 671-682. 1958. [ PubMed ]

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Last updated: April 23, 2013

Pulse Chase Primer: The Meselson-Stahl Experiment

• DNA & RNA
• Experimental Design

Resource Type

Description.

This activity can be used in conjunction with the short film The Double Helix . It introduces students to the classic experiment by Matthew Meselson and Franklin Stahl, which revealed that DNA replication follows the semiconservative model.

In 1958, Meselson and Stahl published the results of a pulse-chase experiment to determine how cells replicate their DNA. Students will first read about how the experiment was conducted and describe the predicted results based on three possible models of DNA replication. They then evaluate the actual experimental results.

Student Learning Targets

Interpret experimental evidence to distinguish between different models of DNA replication.

Describe the semiconservative model of DNA replication.

Estimated Time

conservative replication, dispersive replication, experiment, nucleotide, radioactive, replication, semiconservative replication

Primary Literature

Meselson, Matthew, and Franklin W. Stahl. “The Replication of DNA in Escherichia coli .” Proceedings of the National Academies of Science 44, 7 (1958): 671–682. https://doi.org/10.1073/pnas.44.7.671 .

Terms of Use

The resource is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International license . No rights are granted to use HHMI’s or BioInteractive’s names or logos independent from this Resource or in any derivative works.

Version History

Curriculum connections, ngss (2013).

HS-LS1-1, HS-LS3-1; SEP2, SEP4

AP Biology (2019)

IST-1.M; SP2, SP3, SP4

IB Biology (2016)

Common core (2010).

ELA-RST.9–12.7

Vision and Change (2009)

CC2, CC3; DP1, DP3

Educator Tips

Double Helix and Pulse-Chase Experiment

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The Meselson-Stahl Experiment