PCR: copying DNA in a tube

The polymerase chain reaction (PCR), invented by Kary Mullis (Nobel Prize in Chemistry, 1993), copies a tiny amount of DNA into thousands to millions of copies, in a tube, in a few hours. A machine called a thermocycler heats and cools the tube in repeated cycles.

The ingredients:

• the DNA to be copied (the template);
• two short primers, a forward and a reverse one, which match the DNA on either side of the target;
• the four dNTPs, the building blocks;
• a heat-stable DNA polymerase: Taq polymerase from Thermus aquaticus, a bacterium of hot springs, or the more accurate Pfu from Pyrococcus furiosus.

Each cycle has three steps:

• Denaturation at 94-95 °C: the two DNA strands separate.
• Annealing at about 50-55 °C: the primers bind to their matching sites.
• Extension at about 72 °C: the polymerase builds new strands from the primers.

The number of copies doubles every cycle (2ⁿ after n cycles); 25-30 cycles are usual. The copied pieces, the amplicons, are checked on a gel at the end.

Two important variants:

• RT-PCR starts from mRNA, which is first copied into cDNA by reverse transcriptase.
• Real-time PCR (qPCR) adds fluorescent dyes such as SYBR green, which glow when bound to double-stranded DNA. The amount of product is measured during every cycle, so no gel is needed.

The Covid-19 test is a real-time RT-PCR. It looks for parts of the virus's RNA (the N gene and ORF1a/b), with a human gene, RNase P, as a control to show the sample was taken properly. A probe carries a glowing reporter dye held quiet by a quencher; when Taq cuts the probe, the reporter is freed and glows. The Ct value is the cycle at which the glow crosses a set threshold. The more virus in the sample, the sooner it crosses, so the lower the Ct.