Expansion
Denature, anneal, extend, repeated in cycles
Expansion
Three steps per cycle, typically repeated 25 to 40 times:
- Denaturation at about 95 degrees: the double helix separates
- Annealing at about 50 to 65 degrees: primers bind flanking sequences. The temperature is the critical variable, since too low causes non-specific binding
- Extension at about 72 degrees: Taq polymerase, from a thermophilic bacterium and therefore heat stable, synthesises the new strand
Amplification is exponential: 2 to the power of the cycle number.
Variants
- Reverse transcriptase PCR: converts RNA to complementary DNA first, so it detects RNA viruses and measures gene expression
- Real time (quantitative) PCR: fluorescence measured each cycle, allowing quantification. The cycle threshold is inversely related to the starting amount, so a low value means a high viral load
Requirements: template, primers, polymerase, nucleotides, magnesium and buffer.
Limitations: exquisite sensitivity means contamination is the major practical problem, and a positive result detects nucleic acid rather than viable organism, which is why PCR can remain positive long after an infection has resolved.