Polymerase chain reaction (PCR)

Principle

• PCR amplifies a specific DNA segment exponentially using repeated temperature cycles; ideally copies double each cycle (2^n).
• Two primers (usually 18–25 nucleotides) flank the target; a thermostable DNA polymerase (Taq from Thermus aquaticus) extends them.

Reagents and equipment

• Template DNA, forward and reverse primers, dNTPs, Taq polymerase, reaction buffer and Mg2+ (typically 1.5–2.5 mM; polymerase cofactor).
• Thermal cycler; nuclease-free water; aerosol-resistant tips.

Procedure

• Initial denaturation: 94–95 °C for 2–5 minutes.
• Denaturation: 94–98 °C, 15–30 seconds (strands separate).
• Annealing: about 50–65 °C (usually 3–5 °C below primer Tm), 15–60 seconds.
• Extension: 72 °C, about 1 minute per kb.
• Repeat 25–40 cycles; final extension at 72 °C for 5–10 minutes; hold at 4 °C.
• Detect products on agarose gel with a DNA ladder, or by probes/sequencing.

Results

• A single band of expected size in the test and positive control, with none in the negative control, indicates target presence.

Quality control and pitfalls

• Controls: no-template (negative) control, positive control, and an internal amplification control to detect inhibition.
• Contamination with amplicons is the main risk: one-way workflow with separate pre-PCR and post-PCR areas, dedicated equipment, UNG with dUTP carry-over prevention.
• Inhibitors: heparin, haem, ethanol, phenol, excess EDTA, urea, polysaccharides.
• Low annealing temperature or excess Mg2+ causes non-specific bands; primer dimers appear as small bands below about 100 bp.

Variants

• Multiplex, nested, allele-specific, RT-PCR, real-time PCR, digital PCR.

Clinical use

• Detection of pathogens (TB, HBV, HPV), mutation analysis, HLA typing, forensic identification, paternity testing.