Question

Describe the experiments that helped demonstrate the semiconservative mode of DNA replication.

Answer 1

Messelson and Stahl (1958) cultured bacteria E. coli in a cultural medium containing 15N isotopes 15NH4 Cl (15N is the heavy isotope of nitrogen) of nitrogen. After the replication of DNA of E. coli for many generations in 15N medium, it was found that both strands of DNA contained 15N as the constituent of purines and pyrimidines.

This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient. Being 15N not a radioisotopic isotope, it can be separated from 14N only based on densities.

When these bacteria with incorporated 15N were placed in medium containing 14N (14NH4Cl), it was noticed newly formed DNA molecules contain one Strand heavier than the other. DNA such formed was found to be hybrid as one strand was made up of ‘^N (old) and another was made up of 14N (new). The various samples were separated independently on CsCl gradients to measure densities of DNA after 20 minutes (1st generation). E. coli bacterium divides in 20 minutes. During second replication (after 40 minutes) in the normal 14N medium both the strands again separated (with radioactive and non-radioactive 15N).

It was observed that out of the total four DNA molecules formed two were completely non-radioactive and the remaining two were with one-half radioactive and another half non-radioactive strand.