DNA sequencing Flashcards
What are the five stages in DNA sequencing
1) The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases.
2) The mixture is placed in a thermal cycler (what is used for PCR)
3) At 60ºC, DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single strand DNA template
4) Each time a terminator base in incorporated instead of a base, the DNA strand is halted.
- As the chain terminating bases are in fewer numbers and are added randomly, there is a random sequence of base lengths
- Every possible length of DNA sequence will be produced
- The DNA fragments are then separated according to their length by capillary sequencing which works like gel electrophoresis but in miniature capillary tubes
- The fluorescent markers on the terminator bases are used to identify the final base on each fragment
- Lasers detect the different colours and thus the order of bases
5) The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made.
What advancements are included in high throughput sequencing?
The fragments are in a flow cell rather than a capillary tube which allows millions of DNA fragments to be replicated
