PCR (polymerase chain reaction)

PCR

DNA amplification, creates multiple copies from small samples of DNA

applications

dna research, dna profiling, phylogenetic analysis (specific species containing small/rare amount of samples, shortens time to diagnose genetically rare diseases (amplifies dna to recognise faulty dna)

DNA template molecule

sample of DNA that’s amplified

primers

short synthetic single stranded sections of DNA complementary to target DNA sequence

forward primer binds to the 3’ to 5’ strand, reverse primer binds to 5’ to 3’

nucleotides

used to build new dna strands

taq polymerase

starts off the annealing process

originated from strain of bacteria from hotspring’s (survives to near boiling temps, optimum at 72 degs)

used instead as human dna polymerase breaks down at 95 degrees, which is the temp necessary to separate 2 complementary strands of DNA in thermocycler

buffer

used to create right acidic/alkaline conditions for reactions to occur

thermocycler, pcr tube

temp regulation, holds pcr reaction mixture during amplification within thermal cycler

denaturing

double stranded target DNA heated to approximately 95 degs

DNA unwinds, hydrogen bonds that hold 2 strands tgt weaken and break, forming single strands of DNA

annealing

Cycler reduces to 55C (as primers don’t work at 95 degs, must be lowered to optimum temp), allows forward and reverse primers (on leading, lagging strand), to bind to complimentary base sequences on separated DNA strands

acts as a starting point where replication of new DNA molecules begin

PCR synthesises DNA in the 5’ prime to 3’ prime direction

5’ prime, 3’ prime

on pentose sugars of DNA molecules, there’s 5 carbons. 1 will be exposed (the 5th carbon or the 3rd), which determines which direction DNA is built

elongation/extension

thermocycler increased to 72 to increase rate of elongation

taq polymerase attaches adjacent to primers, reads DNA code and builds complementary strand of DNA (3-5 mins per cycle)