Polymerase chain reaction Flashcards
what is pcr
like a molecular photocopier, is a technique used to amplify very small amounts of dna very quickly
what is the length of dna tthat is usually amplified
100-10,000 base pairs, no longer that 40,000 bp
what are the applications of pcr
parentage testing, forensics, diagnosis of herediatry diseases, gene cloning, genotyping, functional analysis of genes
what is the dna that is desired to be amplifed called
‘the region of interest’
what is needeed for pcr
dna primers that are complementary to the 3’ end of a length of dna to be amplified, thermocycler, taq polymerase, buffer, mgcl2, dna to be amplified and free dna nucleotides
oligo
a short single stand of rna/dna
synthetic dna
dna that is created in a laboratory using a dna synthesiser
thermocycler
a machine capable of ramping between temperatures very quickly, can be programmed to heat/cool dna by cycling between three temps
taq polymerase
dna polymerase from a bacteria thermas aquaticus that lives in geothermal pools and has a high optimal temp
what is the firrst step of pcr
denaturisation - dna is heated to 95°c for 2 minutes, the heat breaks the hydrogen bonds between complementary bases, seperating the two strands in the dna moleecule
what is the second step of pcr
annealing - the mixture is cooled to 55°c for 2 mins, primers added and anneal to the flag sections of dna to be copied
what is a buffer
keeps the ph constant
why is a buffer needed
dna is an acid, so the mixture becomes more acidic and more dna is created, buffer is needed to keep the ph and optimum temp for enzyme
what does denaturing dna mean
it doesn’t mean a permanent alteration as if the dna was cooled, the strands would stiick back togethert
what is the third step of pcr
elongation - mixture is heated to 72°c for 1 min, the taq polymerase adds the free nucleotides via the complementary base pair rule to the single strand of dna being copied, starting from the primar
