VPB 321: Animal Biotechnology (2+1)

STEPS IN AUTOMATED CYCLE SEQUENCING

• There are three major steps in a cycle sequencing reaction (like in PCR), which are repeated for 30 or 40 cycles.
  • Denaturation at 94°C :
    • During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop.
  • Annealing at 50°C :
    • In sequencing reactions, only one primer is used, so there is  only one strand copied in each cycle.
  • Extension at 60°C :
    • This is the ideal working temperature for the polymerase (normally it is 72 °C, but because it has to incorporate ddNTP's which are chemically modified with a fluorescent label, the temperature is lowered.When a ddNTP is incorporated, the extension reaction stops  because a ddNTP contains a H-atom on the 3rd carbon atom (dNTP's contain a OH-atom on that position). Since the ddNTP's are fluorescently labeled, it is possible to detect the colour of the last     base of this fragment on an automated sequencer. Because only one primer is used, only one strand is copied during sequencing, there is a linear increase of the number of copies of  one  strand of the gene.
    • Therefore, there has to be a large amount of copies of the gene  in  the  starting mixture for sequencing.
    • Suppose there are 1000 copies of the wanted gene before the cycling starts, after one cycle, there will be 2000 copies: the 1000 original templates and 1000 complementary strands with each one fluorescent label on the last base, after two cycles, there will be 2000 complementary strands, three cycles will result in 3000 complementary strands and so on.