Main content
Biology library
Course: Biology library > Unit 17
Lesson 3: DNA replication- DNA replication and RNA transcription and translation
- Leading and lagging strands in DNA replication
- Speed and precision of DNA replication
- Molecular structure of DNA
- Molecular mechanism of DNA replication
- Mode of DNA replication: Meselson-Stahl experiment
- DNA proofreading and repair
- Telomeres and telomerase
- DNA replication
© 2023 Khan AcademyTerms of usePrivacy PolicyCookie Notice
Mode of DNA replication: Meselson-Stahl experiment
A key historical experiment that demonstrated the semi-conservative mechanism of DNA replication.
Key points:
- There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive.
- The semi-conservative model, in which each strand of DNA serves as a template to make a new, complementary strand, seemed most likely based on DNA's structure.
- The models were tested by Meselson and Stahl, who labeled the DNA of bacteria across generations using isotopes of nitrogen.
- From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that DNA is replicated semi-conservatively.
Mode of DNA replication
Imagine yourself in 1953, after the double helix structure of DNA has just been discoveredstart superscript, 1, end superscript. What burning questions might be on your mind, and on the minds of other scientists?
One big question concerned DNA replication. The structure of the DNA double helix provided a tantalizing hint about how copying might take placestart superscript, 1, comma, 2, end superscript. It seemed likely that the two complementary strands of the helix might separate during replication, each serving as a template for the construction of a new, matching strand.
But was this actually the case? Spoiler alert: The answer is yes! In this article, we'll look at a famous experiment, sometimes called "the most beautiful experiment in biology," that established the basic mechanism of DNA replication as semi-conservative—that is, as producing DNA molecules containing one new and one old strandcubed.
The three models for DNA replication
There were three basic models for DNA replication that had been proposed by the scientific community after the discovery of DNA's structure. These models are illustrated in the diagram below:
- Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
- Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).
- Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.
Most biologists at the time would likely have put their money on the semi-conservative model. This model made a lot of sense given the structure of the DNA double helix, in which the two DNA strands are perfectly, predictably complementary to one another (where one has a T, the other has an A; where one has a G, the other has a C; and so forth)start superscript, 2, comma, 4, end superscript. This relationship made it easy to imagine each strand acting as a template for the synthesis of a new partner.
However, biology is also full of examples in which the “obvious” solution turns out not to be the correct one. (Protein as the genetic material, anyone?). So, it was key to experimentally determine which model was actually used by cells when they replicated their DNA.
Meselson and Stahl cracked the puzzle
Matt Meselson and Franklin Stahl originally met in the summer of 1954, the year after Watson and Crick published their paper on the structure of DNA. Although the two researchers had different research interests, they became intrigued by the question of DNA replication and decided to team up and take a crack at determining the replication mechanismstart superscript, 5, end superscript.
The Meselson-Stahl experiment
Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system.
They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen, start superscript, 15, end superscript, start text, N, end text. (An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.) When grown on medium containing heavy start superscript, 15, end superscript, start text, N, end text, the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA.
After many generations growing in the start superscript, 15, end superscript, start text, N, end text medium, the nitrogenous bases of the bacteria's DNA were all labeled with heavy start superscript, 15, end superscript, start text, N, end text. Then, the bacteria were switched to medium containing a "light" start superscript, 14, end superscript, start text, N, end text isotope and allowed to grow for several generations. DNA made after the switch would have to be made up of start superscript, 14, end superscript, start text, N, end text, as this would have been the only nitrogen available for DNA synthesis.
Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then measured the density of the DNA (and, indirectly, its start superscript, 15, end superscript, start text, N, end text and start superscript, 14, end superscript, start text, N, end text content) using density gradient centrifugation.
This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube. Density gradient centrifugation allows very small differences—like those between start superscript, 15, end superscript, start text, N, end text- and start superscript, 14, end superscript, start text, N, end text-labeled DNA—to be detected.
Results of the experiment
When DNA from the first four generations of E. coli was analyzed, it produced the pattern of bands shown in the figure below:
What did this result tell Meselson and Stahl? Let's walk through the first few generations, which provide the key information.
Generation 0
DNA isolated from cells at the start of the experiment (“generation 0,” just before the switch to start superscript, 14, end superscript, start text, N, end text medium) produced a single band after centrifugation. This result made sense because the DNA should have contained only heavy start superscript, 15, end superscript, start text, N, end text at that time.
Generation 1
DNA isolated after one generation (one round of DNA replication) also produced a single band when centrifuged. However, this band was higher, intermediate in density between the heavy start superscript, 15, end superscript, start text, N, end text DNA and the light start superscript, 14, end superscript, start text, N, end text DNA.
The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication was a hybrid of light and heavy DNA. This result fit with the dispersive and semi-conservative models, but not with the conservative model.
The conservative model would have predicted two distinct bands in this generation (a band for the heavy original molecule and a band for the light, newly made molecule).
Generation 2
Information from the second generation let Meselson and Stahl determine which of the remaining models (semi-conservative or dispersive) was actually correct.
When second-generation DNA was centrifuged, it produced two bands. One was in the same position as the intermediate band from the first generation, while the second was higher (appeared to be labeled only with start superscript, 14, end superscript, start text, N, end text).
This result told Meselson and Stahl that the DNA was being replicated semi-conservatively. The pattern of two distinct bands—one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-conservative replication (as illustrated in the diagram below). In contrast, in dispersive replication, all the molecules should have bits of old and new DNA, making it impossible to get a "purely light" molecule.
Generations 3 and 4
In the semi-conservative model, each hybrid DNA molecule from the second generation would be expected to give rise to a hybrid molecule and a light molecule in the third generation, while each light DNA molecule would only yield more light molecules.
Thus, over the third and fourth generations, we'd expect the hybrid band to become progressively fainter (because it would represent a smaller fraction of the total DNA) and the light band to become progressively stronger (because it would represent a larger fraction).
As we can see in the figure, Meselson and Stahl saw just this pattern in their results, confirming a semi-conservative replication model.
Conclusion
The experiment done by Meselson and Stahl demonstrated that DNA replicated semi-conservatively, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand.
Although Meselson and Stahl did their experiments in the bacterium E. coli, we know today that semi-conservative DNA replication is a universal mechanism shared by all organisms on planet Earth. Some of your cells are replicating their DNA semi-conservatively right now!
Want to join the conversation?
Why is Cesium chloride used? Why can't the centrifugation be done without it?(18 votes)- The density of the cesium chloride solution increased along a gradient down the tube.(3 votes)
what causes the double helix of the DNA to "unzip"(7 votes)
During DNA replication, the enzyme helicase unwinds the DNA double helix by disrupting the hydrogen bonds that keep it together. Different proteins are also involved in the unzipping of the double helix such as single strand binding proteins that keep the two strands from reforming hydrogen bonds.(24 votes)
- Could someone explain to me the results of the 3rd and 4th generation please? I don't quite understand that part, why isn't all DNA semi-conservative? Why is there a N^14 light strand?(4 votes)
- 1st we know: Bacteria is grown in N15 ,
then placed in N14 .
(remember N14 is the light isotope and N15 is the heavy one.)
So initially all nitrogenous bases of each nucleotide will have contained N15 isotope.
So once place in N14 medium ,DNA replication will use the N14 Isotope only.
No more N15 available.
Thats why in the 3rd and 4th generation , the N15 strand disappears over time
So what remains are DNA Strands that undergo Semi conservative replication with the fixed amount of N15 and an increasing amount of N14, as N14 is available in the medium.
The chart in Fig. 2 kind of explains it.(11 votes)
What would have happened if they grew the bacteria in the light isotope and then introduced it to the heavy isotope? What would the vial layers and densities look like? (Essentially, what would happen if you did the opposite of what they did.)(5 votes)
I think the exact same thing would have happened, but instead of the number of light 14N DNA increasing over successive generations, the heavy 15N DNA would increase with each generation.(3 votes)
when its splitting is there every a case when it doesn't split correctly(2 votes)
Absolutely! There are numerous syndromes, diseases and conditions based on the improper splitting. This is something that is easily googled and you can find tons of information on!(8 votes)
If it is concluded that each strand serves as a template for the synthesis of a new complementary strand, how come a hybrid strand does not produce two hybrid molecules. For example, one light and one heavy template strand, produces a complementary heavy and light strand respectively.(2 votes)
Good question! Keep in mind that the DNA is grown in N15 (heavy) but the next generations are in N14 (light). So a hybrid strand cannot produce hybrid molecules because there aren't N15-labelled nucleotides available! The question and response below might help clarify too :)(4 votes)
Would the results be different if they didn't use E. Coli but something else instead?(3 votes)
No, because DNA replicates semi-conservatively. :)
Maybe the experiment would be set up slightly different but results must be the same.
Replication is semi-conservative in both: Eukaryotes and Prokaryotes meaning that it is the same for any living organism.(2 votes)
It means the other two models don't exist?(1 vote)
Models are proposed explanations for or descriptions of something.
All three of the models discussed in this article "exist", but one of them is a better description of how DNA replication actually occurs.
Does that help?(6 votes)
Why are bacteria like Escherichia coli good for studying DNA replication (as opposed to using other types of cells)?(2 votes)- E. coli is model organism that's why.
Why E. coli model organism?
E. coli is a perfect model for several reasons: E. coli is a single-celled organism that can be manipulated and killed with no ethical concerns. It has a rapid growth rate and is very easy to culture and grow.
https://study.com/academy/lesson/escherichia-coli-e-coli-as-a-model-organism-or-host-cell.html
The main reasons why E. coli is the organism of choice extends and is not limited to its fast growth in chemically defined media; relative cheap culture media; does not form aggregates; industrial scalability; several molecular tools for manipulation; extensive knowledge of its genetics and genomics; extensive knowledge on its transcriptome, proteome, and metabolome, and several strains are considered biosafety 1, which renders it ideal even for teaching and school demonstrations.
In terms of ecology, E. coli is a facultative aerobe (either respiration takes place in the presence of oxygen or fermentation in its absence), which bears a sensor for oxygen presence (redox state in the quinone pool) and can activate or repress the required metabolic enzymes, depending on oxygen levels.
The building blocks of E. coli consists of about 55% protein, 25% nucleic acids, 9% lipids, 6% cell wall, 2.5% glycogen, and 3% other metabolites [16–18], which for biotechnological applications is important since carbon flux is often a problematic issue to address to generate a novel metabolic pathway or to enhance a current functioning pathway.
E. coli is part of the normal microbiota of mammals, rendering the predominant facultative microbe of the gastrointestinal tract and is currently a hot debate on the impact on normal microflora establishment and their role in disease.
E. coli harbours a genome with particular features such as a strikingly organized structure, remnants of many phages, and insertion sequences (IS) and a high transport capacity toward the cytoplasm.
https://www.intechopen.com/books/-i-escherichia-coli-i-recent-advances-on-physiology-pathogenesis-and-biotechnological-applications/-i-escherichia-coli-i-as-a-model-organism-and-its-application-in-biotechnology(2 votes)
what is A, T, G, C in DNA Helix(0 votes)
Those are purines and pyrimidines, those are the nitrogen bases that build DNA molecule. A stands for Adenine, G stands for Guanine, and those two are purines. And pyrimidines are C that stands for Cytosine and T stands for Thymine.(10 votes)