DNA replication and the Meselson-Stahl experiment
The Meselson-Stahl experiment by Matthew Meselson and Franklin Stahl showed that DNA replication was semiconservative.
Semiconservative replication means that the DNA double helix strand, on replication, produces two double-stranded DNA helices. Each one has an original DNA helix strand and one new synthesized DNA helix strand.
In 1953, James Watson and Francis Crick discovered the structure of DNA. Going by the complementary structure of the base sequences of the DNA strands (cytosine base pairing with guanine base, and adenine base pairing with thymine base), they proposed that during DNA replication the old DNA strands would be used in the making of the new DNA strands, resulting in new DNA helices containing one new strand and one old strand. That is, the DNA replication would be semiconservative replication. At that time, there was no conclusive evidence of this.
Matthew Meselson and Franklin Stahl set out to prove this hypothesis in 1958. They considered the semiconservative replication model as well as two other models, the conservative replication model and the dispersive replication model. In conservative DNA replication, two entirely new DNA strands would be produced. In dispersive DNA replication, the old and new DNA would be interspersed with each other along each strand.
The Meselson-Stahl Experiment
To begin with, to be able to tell the old and new DNA apart, Meselson and Stahl grew several generations of <em>Echerichia coli</em> bacteria in two different mediums, one in the “heavy” nitrogen isotope 15N and the other in the “light” nitrogen isotope 14N. So, it turned out, that one bacterial culture contained the heavy form of nitrogen and the other contained the light form of nitrogen.
Then, taking samples of each and extracting DNA into solution, the scientists mixed the two DNA solutions together and added the mix to a CsCl (cesium chloride) solution of the same density. This mix was then processed at high speed in an ultracentrifuge. The result was a mixture separated by density—the “heavy” mix was denser than the “light” mix and so the “heavy” mix sank to the bottom and the “light” mix moved up. Thus it was possible to tell the two apart.
Next, the scientists grew several generations of E.coli in ‘heavy’ nitrogen medium, and extracted a DNA sample from these bacterial cells. They called this sample “generation zero” and prepared it for centrifugation. The result showed that the DNA in generation zero had a heavier density, that is was in the “heavy” form.
The scientists then transferred the E.coli bacterial cells to the “light” nitrogen medium and allowed it to grow in that. They took samples from this every 20 minutes and processed these samples too in an ultracentrifuge.
Results
The first result, after one generation, showed that the DNA had an intermediate density rather than heavy or light. This ruled out conservative replication, as, in that case, the result wouldn’t have shown intermediate density, but rather equal amounts of heavy and light DNA. On the other hand, intermediate density would be possible with both semiconservative and dispersive replication.
The second result, after two generations, showed that one part of the DNA had intermediate density and the other part had light density. This ruled out dispersive replication as in that case the DNA distribution would have been same between the strands and the resulting density would have been lower than the intermediate one.
The third result, after several more generations, showed that now a larger portion of the DNA had light density, synthesized from the first and second generations, and a smaller portion of DNA had intermediate density, sythesized from the heavy and light DNA. This proved the semiconservative hypothesis and was a major step in developmental biology research.
