My friend Anthony Wilson, poet and educator, has published several ‘thin volumes’ of his own wonderful poems and has also produced a beautiful and wide-ranging anthology of other poets’ work, entitled ‘Lifesaving Poems’ (Bloodaxe Books, 2015). In his commentary on Tides, a poem by Hugo Williams, Anthony alludes to the words of Seamus Heaney who became conscious in his late 40s of his need to ‘credit marvels,’ or in the words of another commentator, John Wilson Foster, to nurture ‘a more conscious receptivity to the wondrous inherent in the commonplace’. This is a theme that emerges in Chapter 5 of our book. There I suggested that our familiarity with the mechanisms involved in the working of genes has meant that those mechanisms seem ‘ordinary’ or even commonplace. Indeed, in some ways they are, taking place during every second of every day in every living cell. And yet, if we look beyond that familiarity, that ordinariness, that commonplaceness, they are indeed marvels.
We can see the same sequence of thoughts when we consider how some of the findings of science are used to benefit human society. The announcement that ‘scientists are now able to …’ (fill in your own favourite here) induces our amazement, our wonder but those emotions fade as the particular innovation becomes embedded in medicine/agriculture/IT etc. And thus it has been with PCR – a three-letter abbreviation that has become very familiar to us during the COVID-19 pandemic. However, for many people, that’s where their knowledge of the process stops, i.e., it’s a method used to test for the presence of the virus but there’s no need to know any more than that. However, I want to say more than that because it is a beautiful example of how the knowledge revealed by scientific research (or, as tabloid newspapers might say – the work of back-room boffins) can be put to a use that benefits society.
PCR stands for Polymerase Chain Reaction and those words sum up beautifully the essentials of the process. Let’s start at the beginning. A polymerase is an enzyme (a biochemical catalyst) that makes polymers – molecules built up from many similar smaller molecules. As we describe in Chapter 5, DNA is a polymer and the individual units of which it is made are called bases (1). Enzymes that build DNA are called DNA polymerases (2). The American biochemist Kary Mullis proposed that the stable DNA polymerases from certain heat-tolerant bacteria could be used in a ‘chain reaction’ to amplify (make many copies of) pieces of DNA (3). He successfully demonstrated the technique in December 1985 and it quickly became embedded as a routine tool in molecular biology. Mullis shared the 1993 Nobel Prize for Chemistry in recognition of the importance of this.
Further, we can, in any sample of DNA, direct the reaction so that it only copies the segments that we want to be copied. The detail is not important except to say that this is achieved by use of primer molecules that can be synthesised in the lab to recognise particular DNA sequences. PCR will then amplify the tract of DNA between the two primers. We can build up a stock of any gene or other DNA sequence in which we are interested. Further, as I am sure dear reader you will have realised, if the sequences at which the primers are aimed are not actually present in our DNA sample, then no amplification occurs. The importance of this is immediately apparent when we apply the technique to the detection or otherwise of, for example virus genes (more about this later in this blog).
As mentioned above, it did not take long for PCR to become established as a routine tool in labs working on genes, including my own at the University of Exeter. Prior to the technique’s invention, amplification of particular genes or other sequences was done by growing them in bacteria by a process called molecular cloning. It was certainly more cumbersome and time-consuming than PCR and it was often difficult to clone just the piece of DNA in which one was interested. The small machines (with a footprint smaller than a piece of A4 paper) in which PCR is carried out became part of the standard kit in any molecular biology or molecular genetics lab. The wondrous had become commonplace.
At this point a quick reminder of basic mechanisms involved in the way that genes work will help to understand how PCR has been used during the pandemic. When a gene is working, it is not the code in the DNA itself that is read but the code in a copy of that gene. That copy is not made of DNA but of RNA and is known as messenger RNA (mRNA), as described more fully in Chapter 5. However, some viruses, including SARS-CoV-2 (the virus that causes COVID-19) use RNA as their genetic material. It is the genes themselves that act as mRNA, without the need to copy from a DNA template (4) and the unsuspecting host cells use that virus mRNA to make components of the virus.
If you have followed this discussion so far, you can see that this might be a problem for PCR, which amplifies DNA, not RNA. Once again however, scientific discoveries come to our rescue. There are some viruses whose ‘life-style’ involves copying RNA into DNA. These include HIV and, in the plant world, cauliflower mosaic virus (CaMV). And of course, there is an enzyme (biochemical catalyst) that does this. It is called reverse transcriptase (RT), a DNA polymerase that makes DNA copies of RNA molecules (5). When we use it in the lab we can again employ specific primers (see above) so that only the RNA of interest is copied. This can then be amplified by conventional PCR which, by using careful changes of conditions, takes place in the same tiny test tube as the RT reaction.
Virus RNA (or a specific section of virus RNA) is copied into DNA with RT; DNA is amplified by PCR.
In testing for SARS-Cov-2, a standard is included to make sure the technique is working and the technician will look to see whether or not there has been any amplification of virus-specific sequences. A lack of amplified viral sequences indicates an absence of virus in the original sample, i.e., a negative test result.
There are different ways of detecting the amplified DNA. In one method, fluorescent DNA-binding chemicals are used and in larger, more sophisticated PCR machines than the basic machines I mentioned earlier, the detection of the fluorescence can be done in situ as the reaction proceeds. With suitable calibration with appropriate standards, the amount of fluorescence can be related to the amount of RNA in the original sample. In other words, the process can be quantitative – hence the shorthand name for the overall procedure is qRT-PCR. In my lab for example, we used this procedure to compare the amounts of mRNA from two different genes in dividing plant cells (6).
So, if my descriptions have been clear enough – and I hope that they have – next time you hear about a PCR test for presence versus absence of SARS-Cov-2, I hope you’ll think about Seamus Heaney looking to ‘credit marvels’ in the commonplace. I hope you’ll remember the skill and inventiveness of Kary Mullis and of the scientists who subsequently tweaked his PCR technique. I hope that you will see the marvels in the ‘commonplace’ but nevertheless beautiful biochemical reactions, reactions that are going on all around us – and indeed within us – that are employed in the PCR procedures. I hope you will see the ‘poetry of nature’ in those reactions. And dare I hope that you’ll also thank God for science?
(1) Technically, the building blocks are actually deoxyribonucleotides; a base is part of a deoxyribonucleotide (or, in RNA, part of a ribonucleotide).
(2) These are enzymes with which I and my research team are very familiar: e.g., https://doi.org/10.1093/jxb/43.1.31
(3) When working optimally, the process can generate a billion copies from a single piece of DNA in just a few hours.
(4) Tobacco mosaic virus (which also attacks tomato plants) also has an RNA genome which acts directly as mRNA (or in technical terms, is a + stranded RNA virus). It was widely used in research as a ‘model’ for viruses of this type, including our studies of how the viral genome is copied: https://doi.org/10.1093/oxfordjournals.aob.a086791, https://doi.org/10.1093/oxfordjournals.aob.a086933
(5) It was a privilege for me to be part of the group that discovered and started to characterise the CaMV reverse transcriptase https://doi.org/10.1093/nar/13.12.4557