The Meselson-Stahl experiment

• what did Meselson and Stahl do?

  they did an experiment on DNA replication
• what is the 1st part of the Meselson-Stahl experiment?

  • they carried out an experiment in which they cultured the bacterium (Escherichia coli) for several generations in a medium containing amino acids made with the heavy isotope of nitrogen, 15N, instead of the normal light isotope, 14N
  • the bacteria incorporated the 15N into their nucleotides and then into their DNA so that eventually, the DNA contained only 15N
  • they extracted the bacterial DNA and centrifuged it
  • the DNA settled at a low point in the tube because the 15N made it heavy
• what is the 2nd part of the Meselson-Stahl experiment?

  • the 15N bacteria were washed, then transferred to a medium containing the lighter isotope of nitrogen, 14N, and were allowed to divide once more
  • the washing prevented the contamination of the 14N medium with 15N, so that 15N was not incorporated into any new DNA strands
• what were the results from the first generation of DNA in the Menelson-Stahl experiment?

  • DNA from this first generation culture was centrifuged, and had a mid-point density
  • this ruled out conservative replication, as that would produce a band showing the parental molecule that was entirely heavy
  • the intermediate position could imply one strand of the new DNA molecule was an original strand of 15N DNA and the other half was newly made, with 14N, as in semi-conservative replication or it could imply that all strands contained a mixture of light and heavy, as in dispersive replication
• what were the results from the second generation of DNA in the Meneslon-Stahl experiment?

  • DNA from the second generation grown in 14N, settled at the mid-point and high point in the tube, in equal amounts
  • the sample at the mid-point had intermediate density and the sample at the high point was light, containing nitrogen that was 14N only
  • this rules out dispersive replication as, if that were the case, there would always be a mixture of light and heavy in every strand and one band only would form
  • one parental strand is conserved, so this is conclusive evidence for semi-conservative hypothesis
• how can you see the DNA bands?

  they aren’t visible with the naked eye, but can be seen when they are made to fluoresce, with ultra-violet light
• what does this diagram show?

  the stages within semi-conservative replication

  
• what does the enzyme DNA polymerase need to replicate DNA?

  • single-stranded DNA, as a template
  • the four nucleotides, each containing a deoxyribose an the base A, G, C or T
  • ATP, to provide energy for synthesis
• what is the first stage of semi-conservative DNA replication?

  having a DNA double helix
• what is the second stage of semi-conservative DNA replication?

  • helicase breaks the hydrogen bonds holding the base pairs together
  • DNA unwinds/unzips, catalysed by the enzyme helicase and the 2 strands of the molecule separate
• what is the third stage of semi-conservative DNA replication?

  • the enzyme DNA polymerase catalyses the condensation reaction between the 5’-phosphate group of a free nucleotide to the 3’-OH on the growing DNA chain
  • each chain acts as a template and free nucleotides are joined to their complimentary bases
• what is the fourth stage of semi-conservative DNA replication?

  • there are newly synthesised daughter strands
  • they carry bases complimentary to the bases on the other strands(different colour to the daughter strands on diagrams), which acted as the templates for their synthesis
• when DNA replicates, what direction are the template strands read by via DNA polymerase?

  in the 3’-5’ direction
• what direction are the new strands synthesised in and why?

  5'-3'due to the directionality of DNA polymerase and the antiparallel nature of DNA strands (polynucleotide chains)