What is DNA sequencing?
-technique where we can map out the base sequence of an individuals genome
What are the 5 things needed for DNA sequencing?
- DNA sample to be sequenced
- free nucleotides in excess
- coloured fluorescent-labelled terminator bases
- DNA polymerase
- DNA primers
What are the 4 steps to sequence a DNA strand?
- heat up strand to 95 to denature DNA = 2 helixes
- cool down to 50 - primers anneal
- heat to 60 - DNA polymerase joins up nucleotides = new strand - but terminators present
- electrophoresis or laser box
What does electrophoresis do during DNA sequencing?
-direct UV light onto nylon membrane
-smaller = furthest = start of gene
What happens during the laser box in DNA sequencing?
-shines laser as bases pass through
-produces the long stretch of bases
What are terminator bases?
-have a H instead of a hydroxyl group at 3’ carbon of ribose
-can’t form phosphodiester bonds to the next
What are the 2 other types of DNA sequencing?
- nanopore
- pyrosequencing
What is nanopore sequencing?
-DNA strand is unwound through a protein channel
-causes a current that varies depending on the base
-computer can decode currents into sequences
What is pyrosequencing?
-different bases are added individually and then removed
What is an example of pyrosequencing?
-polymerase waits for a T
-when added the anime is active
-triggers luciferase = produces a flash that can be detected electronically
-2x lots = double the brightness
