pcr

steps of PCR

1. denaturation at 90 degrees

2. annealing at 50 degrees

3. extension at 72 degrees in the 5’→ 3’ direction

• primers anneal to the separated strands in opposite direction and serve as starting points for replication by Taq

when does extension stop

• where there is no more template (telomere in genomic dna)

• when there are no more dNTPs (unlikely due to excess)

• when the temperature switches back to 95 degrees

should the amplicon contain the primer sequences

yes

what is the band seen on the agarose gel

• amplicon

• dna fragments are too dilute to appear on the gel

equipment needed for pcr and finding polymorphism

• agarose gel electrophoresis kit

• bench centrifuge

• blue and yellow tips

• ethidium bromide

• pipette

• microfuge tubes

• pcr tubes

• restriction enzymes

• thermocycler

• universal tubes

polymorphic site

site in the genome where the dna sequence is known to be variable among individuals

example of single nucleotide polymorphism

• sickle cell anaemia

short tandem repeat example

• huntingtons disease

transpopsons

• jumping genes

• segments which move from one location to another

• in gel electrophoresis will show larger band for inclusion (and maybe not for hetero)

how is snp followed through for gel electrophoresis

• pcr

• followed by restriction fragment length polymorphism RFLP (uses restriction enzymes)

what should largest fragment from snp polymorphism be equal to

• sum of the two smallest fragments