DNA sequencing

• DNA sequencing is where you understand and map the order of bases in a DNA molecule.
• What is a terminator nucleotide?

  • Modified so that phosphodiester bonds cannot be formed.
  • It can bind to the next nucleotide in the chain but it won’t let others bind after it has. This means DNA synthesis is stopped.
• DNA sequencing principles:

  1. Separate the DNA you want sequenced into four tubes
  2. Each tube contains DNA polymerase, an excess of free nucleotides, some primer.
  3. In one tube you put a low concentration of radioactively labelled terminator base A, in the next you put radioactively labelled terminator base G etc etc.
  4. Run the reaction like PCR.
  5. Do gel electrophoresis to separate the fragments by size.
• DNA sequencing principles:

  • Incorporation of nucleotides is random.
  • This means there will be lots of fragments ending with a A in the first tube. Lots ending with G in the second etc.
  • If the length of the fragment is known (by gel electrophoresis) then you can estimate the location of the bases in the sequence.
• Modern innovations:

  • Computers and computing power.
  • Use UV fluorescing tags rather than radioactive tags and X-rays
  • Capillary sequencing is used rather than gel electrophoresis.
• Modern innovations:

  • Massive parallel sequencing or next generation sequencing updates the process further by conducting all the PCR and imaging on one slide called a flow cell.   
• Phylogeny = evolutionary history of an organism.

  • DNA sequencing allows us to map evolutionary relationships between organisms.
  • It can also indicate how long ago they diverged.
• Classification

  • New organisms can be classified more easily and added to a common database.
  • Food testing – check the food is what the packet says it is.
  • Captive breeding – match animals with genetically different partners to increase genetic diversity. The converse would allow inbreeding.
• • In medicine you can map the genome of a human and look for patterns between diseases and specific base sequences. E.g.
  • The gene responsible for DMD (muscular dystrophy) was found in 1986.
  • If you have the BRCA1 or BRCA2 gene mutation you are more likely to get breast cancer
• Mapping the genome of pathogens allows you to know how fast it is mutating, what those mutations mean for virulence and transmissibility and therefore what counter measures you should take.
• Why DNA sequencing doesn’t provide all the answers…

  • Most traits are polygenic.
  • Epigenetics plays a large role in expression of a gene e.g. spliceosomes.
  • There is not a 1:1 ratio between genes and the proteins they produce