DNA sequencing

• describe how DNA sequencing has changed over time

  • DNA sequencing techniques refined as new technology is developed
  • Sanger sequencing- radioactive labelling of bases and capillary sequencing
  • carried out manually
  • time consuming
  • high throughput techniques developed which are faster eg- massively parallel sequencing where clusters are sequenced and imaged at the same time
  • automated
  • cheaper so more genomes can be sequenced
  • sequencing now takes place on flow cell instead of gel or capillaries
• why is capillary sequencing useful?

  • allows DNA sequences to be produced of entire genomes
  • all possible DNA chains produced with the reaction stopping at every base
  • at the end, fragments are compared so a genome is put together
  • scientists can identify the genes or parts of the genome that are responsible for specific characteristics
  • can identify regions which are linked to particular diseases
• state the stages of DNA sequencing using the capillary (Sanger) method

  1. DNA for sequencing is mixed with a primer, polymerase, excess of normal, complementary nucleotides and terminating nucleotides
  2. put in thermocycler. At 90° the strands denature into single strands, at 50° primers anneal
  3. At 60° polymerase binds primer to DNA from 5' end, forms H bonds bet bases. Polymerase synthesises new strand by forming H bonds bet a normal nucleotide and complementary one
  4. terminator nucleotide is added, chain is terminated different lengths of DNA produced as terminating nucleotides present at smaller amounts + added at random times
• how are the resulting DNA chains ordered by length after the sequencing process has finished?

  • capillary sequencing is used
  • similar to electrophoresis, fluorescent markers on each base used to identify final base on each fragment. Lasers detect different colours and the order of the sequence
  • resolution of the sequencing machine is high enough to register each base in the sequence
• how are terminating nucleotides formed?

  removal of hydroxyl group from a base so it can't form phosphodiester bonds with another base
• why is DNA sequencing useful?

  • use in forensics in terms of catching criminals
  • to find out the likelihood of offspring developing diseases
  • analysis of disease
  • maternity/paternity testing
• what does DNA sequencing involve?

  the sequencing of entire genomes