Polymerase Chain Reaction

PCR

method of recreating in vivo replication of DNA in an in vitro setting

equation for determining number of copies of DNA amplified in PCR

2ⁿ

with n being the number of cycles

amplicon

the copies of your target sequence that are replicated at the end of PCR

master mix components

• DNA template

• oligo primers

• nucleotides

• polymerase

• buffers

how much DNA template is needed for master mix?

10²-10⁵ copies

how much oligo primer is needed for master mix?

0.25 mM of each primer

how much nucleotides are needed in master mix?

0.2 mM of each dNTP

how much polymerase is needed in master mix?

2.5 units

how much buffer is needed in master mix?

10 mM of Tris and 1.5 mM of MgCl₂ (co-factor for polymerases)

primers are _____ probes

not the same as

• probes are used in quantification and can amplify and detect in the same assay

forward primer

hybridize to the target 5’ end

reverse primer

hybridizes to the target 3’ end

target sequence

sequence that is to be amplified

primers flank—

the target sequence

Tm (melting temperature)

temperature at which the DNA begins to denature

Tm equation

Tm= 4^oC (G-C) + 2^oC (A-T)

Taq polymerase

heat stable polymerase that was isolated from Thermus aquaticus

what happens if you vortex the polymerase?

it denatures

PCR buffer function

provides optimal condition for enzyme activity

MgCl₂ and KCl buffers provide—

mono and divalent cations which are important in annealing and enzyme activity

Tris buffer maintains the proper pH of—

8-9.5

BSA (bovine serum albumin)

binds inhibitors and stabilizes the enzyme

thermocycling steps

denaturation, annealing, extension

denaturation

makes both strands accessible by heating to 90-96^oC for 20-60 seconds

annealing

two oligo primers anneal (hybridize) to the complementary strand at 50-70^oC for 10-60 seconds

extension

polymerase synthesizes copies of template by adding nucleotides at 65-75^oC for 10-60 seconds

why are more than 35 PCR cycles not recommended?

the Taq polymerase becomes less effective with each cycle, more cycles exhaust the master mix components

positive control for PCR

makes sure PCR function and specifically functions for the target sequence (spiking with target DNA)

negative control

ensures that there is no contamination within the run (water sample)

internal control

ensures that EACH individual sample that is negative is true negative (spiking with unrelated DNA)