DNA Sequencing and Amplification

  • DNA can be sequenced by replicating with a dideoxynucleotide triphosphate – that is a deoxynucleotide triphosphate with no OH group on Carbon 3 of the sugar. This is where a phosphate group would normally bind as part of a DNA backbone – and as this is no longeran option the replication stops when this dideoxynucleotide is added.
    (The small difference between deoxynucleotides and dideoxynucleotides)
  • I’ll try to explain further. If you were looking for all the adenine positions in a DNA strand, you would add ddATP (dideoxyadenine triphosphate) which would cut the replicated strands in different adenine positions.

3′-AGTTCAATGGCATTAT
5′-TCAAGTTACCGTAATA (correct, using dATP)
—- Possible Outcomes —-
5′-TCA
5′-TCAA
5′-TCAAGTTA
5′-TCAAGTTACCGTA
5′-TCAAGTTACCGTAA
5′-TCAAGTTACCGTAATA

This would leave you with a DNA mixture containing different size DNA fragments, all cut after an Adenine residue. To assess the location of these cuts:

  • Denature the dsDNA (double stranded DNA) – this unpairs the new (fragmented) strands from the old DNA strand.
  • Seperate the DNA by polyacrylamide gel electrophoresis (or use agarose gel).
  • Smaller DNA fragments will travel further, faster than larger DNA fragments, and these fragments will be visible after the addition of a florescent chemical.

Now you know the location of Adenine bases, you can repeat the above with ddGTP, ddTTP or ddCTP, revealing the locations of those bases. You could then compare the seperate gels to work out the DNA sequence. To compare, you would need to run on the same gel:

The -ve control would either be clean or just the original DNA strand – to be used as a reference. The +ve would contain a mix of all of the mixtures.

Then it’s a simple case of looking through the different bands. Remember that towards the top are larger fragments and smaller fragments at the bottom – so you read the sequence from the bottom.

CACTCAGTGATG – and the final top strand is the full DNA strand.

– Amplifying DNA – PCR

To amplify a sample of DNA (polymerase chain reaction):

  1. Denature the double stranded DNA sample to leave single stranded DNA (heat).
  2. Add short primers that a complementary to the ends of the sequences of interest.
  3. Lower temperature and anneal.
  4. Use a thermostable DNA polymerase (a polymerase stable under heat) such as Taq polymerase to extend from the primers.
  5. Denature sample again and repeat from 3.

Repeated, this produces exponential amplification of a DNA sample – eg 40 repeats gives 2^40 amplification! This is assuming good conditions with thermostable DNA polymerase and presence of enough dNTP’s (deoxynucleotide triphosphates) and RNA primers.