Polymerase chain reaction

PCR

duplicates DNA, 30 cycles

2^n

DNA polymerase + primer

Primer

Specificity of reaction, starting position for sequence

5’—> 3’

Probe

used in quantification assays which amplify and detect same assay

Taq polymerase

heat stable, Thermus aquaticus, DNA polymerase

PCR buffers

Optimal conditions for enzyme activity

MgCl2, Tris, BSA

MgCl2 buffer

divalent cations affect primer annealing, increase enzyme activity

Tris buffer

maintains proper pH for enzymes 8-9.5

BSA buffer

Bovine Serum Albumin, binds inhibitors and stabilizes the enzyme

Master Mix Preparation

includes buffer, enzyme, dNTP

Keep in COLD

Thermocylcer

Denaturation

Annealing

Extension

Denaturation

Opens double helix at 90-96 degrees

Annealing

PCR specificity, two oligo primers (forward and reverse) anneal to complementary sequence

50-70 degrees

Extension

elongation, primers synthesize DNA

Polymerase enzymes add nucleotides

65-75 degrees

Positive controls

ensure enzyme is active, buffer optimal, primers are correct

Negative controls

Ensures reaction is not contaminated with amplified products

Internal controls

Contains set pf primers and an unrelated target, ensure reaction is working w/o test sample

Distinguish true negative and internal control failure

UNG amperase

Uracil N Glycosylase

Can specifically degrade products that have been through PCR, leaving native nucleic acid template.

Reverses incorrect amplification