PCR was an acronym that dominated the news in 2020. What is the technique, how useful is it, and what does it tell us?
Polymerase Chain Reaction refers to a technique that makes a large volume of DNA or RNA from a small sample. This is a standard technique in molecular biology that has been used for forensics and DNA fingerprinting, sequencing the human genome, identifying and developing good crops, building evolutionary family trees, prenatal screening, and, more recently, for detecting SARS-CoV-2.
Just as a thermometer is the standard measuring tool for temperature, PCR is the standard technique for identifying and amplifying DNA and RNA.
The technique takes small pieces of DNA or RNA and makes millions of copies that can be detected, sequenced, cloned or diagnosed.
If you had thousands of grains of a substance, you would be able to tell if it’s sand, or sugar. But imagine trying to identify something if you only had one grain. Likewise, small amounts of DNA are hard to identify, but large quantities are easy.
What PCR is looking for
Let’s first establish what DNA and RNA are.
Deoxyribonucleic acid is a long chain polymer (molecule) make of small units called nucleotides. It is a double helix make of two strands that are joined together by complimentary (opposite but bonded) nucleotides that are unique to each person.
RNA is also a polymer made of nucleotides, but it is only a single strand. Instead of DNA, some viruses have RNA that is unique to them. It is more fragile than DNA, and can’t be used for all diagnostic purposes.
Thankfully, PCR is accurate, fast and sensitive, and can amply both DNA and RNA.
How PCR works
First, a small sample of DNA or RNA is taken. This could be from blood, spit, skin or even leaf tissue. Almost any cell that has RNA or DNA can be used, but some are harder to use than others.
For a COVID-19 test, this sample will come from saliva or mucus, which will contain small viral particles with RNA if the person is or was infected.
This is called the template – it is only in very small amounts and cannot be detected on its own.
Next a primer is added. This is a small unit that has nucleotides very specific to the virus that is being tested. If the primer detects the right virus, it will attach to it and recruit a polymerase. This is an enzyme that ‘builds’ polymers – in this case, DNA and RNA – out of spare nucleotides.
For RNA, an extra step is added that turns the RNA into DNA for the rest of the process, so the whole PCR is referred to as reverse transcriptase PCR (RT-PCR). This is useful for preserving the fragile RNA.
With the spare nucleotides and primer, the polymerase can build another copy of the DNA. After one cycle of this, it repeats, so now two DNA copies become four, and four become 8 and so on until there are millions of copies of the DNA. This all happens in a machine called a thermocycler.
Millions of copies of DNA are much easier to detect than one or two, just like a handful of sand is easier to see than a single grain.
Two things can happen after this: The DNA/RNA could be sequenced in further tests to check for new mutations, or the SARS-CoV-2 sequences could glow.
In the latter case, this happens because another chemical is added to the mix. These fluorescent light ‘signals’ are designed to attach to SARS-CoV-2 sequences but not other types of DNA/RNA, and they will glow under the right type of light (usually UV).
If the sample glows, the test result is positive, and if the sample is present but doesn’t glow, the test sample is negative. If there is no sample at all, the PCR failed.
Three cycles of PCR
- Denaturation – The DNA double helix bonds are broken so that it opens up into single strands.
- Annealing – The primers attach the the single stranded DNA.
- Extension – The polymerase reads the open DNA and builds a complementary chain that joins the the existing nucleotides. At the end of the cycle, the DNA is double stranded again.
How effective is PCR?
PCR was first invented in 1985 and is a well-established, common, standard laboratory practice for molecular biology, genetics and medical diagnostics. PCR is highly accurate and sensitive and is considered the gold standard DNA/RNA identification and SARS-CoV-2 diagnosis.
The technique is so accurate at building the right DNA strand that it is used to build DNA for use in CRISPR and other cloning techniques.
Occasionally, false negatives or positives will arise. Regardless, the rates are extremely low and usually happen because of a low quality or old sample – the problem is the sample collection, rather than the test itself. One study of PCR SARS-CoV-2 tests found that just 5 patients in 96,000 came back with a false negative result.
Thankfully, it is usually easy to flag which results might be a false negative because low quality samples are visible to experts, and they are able to retest the sample. This means that the actual number of false negatives upon COVID diagnosis is even lower.
The test is also very sensitive and only needs tiny volumes of sample, such as what is on a swab, where other techniques need a higher volume (e.g. blood).
It is also a relatively quick test. It only takes a couple of hours to run, and multiple samples can be run together.
The speed, ease, sensitivity, and accuracy of PCR is very fine-tuned, and is therefore an unshakable standard in the world of molecular biology. It has been used for decades and will continue to be used for decades to come.
Debbie has run more PCRs that she has visited thrift stores and that is saying a lot.
Dr Deborah Devis is a science journalist at The Royal Institution of Australia.
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