How did they distinguished the heavy DNA molecule from the light DNA molecule ?
Answers
*They grew E.coli in 15NH4ClNH4Cl medium for many generations. (15N is heavy nitrogen not radioactive element)
* The result was that 15N was incorporated into newly synthesized DNA and other nitrogen containing compound as well.
* This heavy DNA molecule could be distinguished from normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
* Then they transferred the E.coli into a medium with normal 14NH4ClNH4Cl and let them grow.(E.coli divides in 20 minutes)
* They took samples at definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices.
* Various samples were separated independently on CsCl gradients to measure the densities of DNA.
* The DNA that was extracted from the culture one generation after the transfer from 15N to 14N medium had a hybrid or intermediate density.
* DNA extracted from the culture after another generation (after 40 min.) was composed of equal amount of this hybrid DNA and of 'light' DNA.
Explanation:
Circular molecules of DNA, such as plasmids and typical mitochondrial genomes, consist of two strands of DNA called the heavy strand (or H-strand) and the light strand (or L-strand). The two strands have different masses due to different proportions of heavier nucleotides.