6.4.1 The Experimental Proof

It is now proven that DNA replicates semiconservatively. It was shown first in Escherichia coli and subsequently in higher organisms, such as plants and human cells. Matthew Meselson and Franklin Stahl performed the following experiment in 1958:

  • They grew E. coli in a medium containing 15NH4Cl (15N is the heavy isotope of nitrogen) as the only nitrogen source for many generations. The result was that 15N was incorporated into newly synthesised DNA (as well as other nitrogen containing compounds). This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient (Please note that 15N is not a radioactive isotope, and it can be separated from 14N only based on densities).
  • Then they transferred the cells into a medium with normal 14NH4Cl and took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices. The various samples were separated independently on CsCl gradients to measure the densities of DNA. Can you recall what centrifugal force is, and think why a molecule with higher mass/density would sediment faster?
  • Thus, the DNA that was extracted from the culture one generation after the transfer from 15N to 14N medium [that is after 20 minutes; E. coli divides in 20 minutes] had a hybrid or intermediate density. DNA extracted from the culture after another generation [that is after 40 minutes, II generation] was composed of equal amounts of this hybrid DNA and of ‘light’ DNA.

If E. Coli was allowed to grow for 80 minutes then what would be the proportions of light and hybrid densities DNA molecule? Very similar experiments involving use of radioactive thymidine to detect distribution of newly synthesised DNA in the chromosomes was performed on Vicia faba (faba beans) by Taylor and colleagues in 1958. The experiments proved that the DNA in chromosomes also replicate semiconservatively.

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