Experimental Evidence for Semi-Conservative Replication

• Which pair of scientists proved that DNA replication was semi-conservative?

  Meselson and Stahl
• Facts that Meselson and Stahl based their work on:

  • ALL the bases in DNA contain nitrogen
• Facts that Meselson and Stahl based their work on:

  • Nitrogen has two forms: the lighter nitrogen 14N and the isotope 15N, which is heavier
• Facts that Meselson and Stahl based their work on:

  • Bacteria will incorporate nitrogen from their growing medium into any new DNA that they make
• The semi-conservative model proposed:

  • The original DNA molecule split into two separate strands, each of which replicated its mirror image (i.e the missing half)
  • Each of the two new molecules would therefore have one strand of new material and one strand of original material
• DNA samples made with nucleotides containing the different isotopes of Nitrogen can be separated by their weight into a centrifuge

  • 'Heavy' DNA (15N) will sink
  • 'Light' DNA will settle out higher up in the solution

  
• Meselson and Stahl's experiment: Step 1

  • To get samples of 'heavy' and 'light' DNA, they grew two cultures of bacteria in nutrient broth that contained either light or heavy nitrogen so that it would be incorporated into their nucleotides
• Meselson and Stahl's experiment: Step 2

  • The bacteria grown with the heavy 15N nitrogen were then put into a broth containing only light 14N nitrogen
  • This meant that when they replicated, the parent or original DNA strand would contain the heavy nucleotides, but to make the new DNA strands they would only be able to use light nucleotides
• Meselson and Stahl's experiment: Step 3

  They took a sample after one generation, and found:

  • The band settled half way between the heavy and light samples, so was semi-conservative
• Meselson and Stahl's experiment: If semi-conservative:

  • All the DNA would contain a mixture of heavy and light DNA as there would be one strand of each in the double helix
  • AND the band would settle half way between the heavy and light samples
  • This is what they found!