DNA sequencing

• DNA sequencing= determining base pair sequence of DNA/ finding the order of nucleotide bases.
• Sequencing

  • Whole genome is too large
  • DNA is copied
  • Copied DNA is cut into fragments
  • Fragments are sequenced separately
  • Fragments are puzzled back together.
• DNA sequencing

  1. DNA for sequencing is mixed with a primer, DNA polymerase, excess of nucleotides and terminator bases.
  2. Mixture is placed in a thermal cycler that rapidly changes temperature at intervals (PCR)
  3. At 60 degrees the DNA polymerase starts to build new DNA strands by adding nucleotides with complementary bases to the single strand DNA template.
  4. Terminator bases are randomly added, stopping DNA synthesis, resulting in DNA fragments of different lengths.
  5. DNA fragments are put in order to length using gel electrophoresis, fluorescent tags are used to identify final base.
• Terminator bases= modified versions of the four nucleotide bases (A,T,C,G) which stop DNA synthesis.

  • A terminator bases will stop DNA synthesis at the location each base would go.
  • They are also given a fluorescent tags.
• Next generation sequencing

  • Fragments are replicated using PCR producing clusters of identical DNA, and are then sequenced= massively parallel sequencing.
  • Thousands of fragments can be sequenced at once.