The current Covid-19 pandemic has highlighted the need for rapid and accurate quantitative analysis of dangerous pathogens, particularly Sars-Cov-2. Fortunately, our ability to determine the structure of new and dangerous viruses has continued to improve since the invention of polymerase chain reaction (PCR), which enables the production of billions of copies of a single DNA sample.
The ‘gold standard’ for DNA detection today is quantitative polymerase chain reaction (qPCR), which combines standard PCR with dye molecules whose fluorescent output increases as the reaction proceeds. This optical monitoring of the PCR reaction in real time – in minutes rather than days – has revolutionised PCR based detection of DNA.
qPCR is based on a multistep thermal cycle (Figure 1). Each cycle, which can take less than a minute, results in a doubling of DNA. Step one separates the sample DNA into single strands by raising the temperature to >90°C, which breaks the hydrogen bonds holding the double stranded DNA together. Step two is an annealing step where the temperature is lowered (55 to 60°C) so that short DNA sequences called primers and probes can attach to the single-stranded DNA. Step three is an extension step where the polymerase enzyme builds the two single strands of DNA into two double strands. Note that although the Sars-Cov-2 virus is RNA based, reverse transcription is used to convert the RNA to complementary DNA before qPCR testing begins.