Polymerase chain reactionBMJ 1997; 314 doi: https://doi.org/10.1136/bmj.314.7073.5 (Published 04 January 1997) Cite this as: BMJ 1997;314:5
Identifies genes and infectious agents
- D A J Tyrrell, Former director of the MRC Common Cold Unita
The polymerase chain reaction (PCR) was devised just over a decade ago, yet it is already an integral part of much biological and medical research. A glance at current journal articles shows that it is also being used to develop new diagnostic tests, which are already having an impact on clinical practice. So it is important for doctors to know the principles on which new tests are based, some of the different versions of the method, and the uses to which they are put.
The polymerase chain reaction is a way of “amplifying” or making multiple copies of any desired piece of nucleic acid. It was first used to make copies of all or part of the DNA of genes. 1 shows the principal steps in the procedure. Firstly, a double strand of DNA is separated into two single strands by heat. Secondly, two rows of nucleotides are marked or “primed” by the addition of two short strands–oligonucleotides–designed to bind specifically on either side of the section of interest in the gene. Thirdly, a polymerase enzyme synthesises a copy of the nucleotide sequence between the primers in the form of a new double strand. Fourthly, the process is repeated and at each stage the number of copies is doubled–from two to four to eight and so on. This can be done quite simply because all the reagents can be added to one tube and the reactions controlled by changing the temperature (the first reaction at 94°C, the second at 55°C, and the third at 72°C using a special heat stable Taq polymerase). As a cycle takes only a few minutes it is possible to generate millions of copies of the DNA in a day.
RNA can also be studied by making a DNA copy of the RNA using the virus enzyme reverse transcriptase. This approach allows us to study messenger RNA (mRNA) in cells that are using the molecule to synthesise specific proteins or for detecting the genome of RNA viruses. Originally, unstable and toxic reagents had to be used, but this can now be avoided.
This technology has transformed the way many molecular studies are done. For example, if you want to determine whether a gene with a particular sequence is present, the polymerase chain reaction will amplify it and other tests can identify it.1 2 If you want to determine whether a gene is directing a protein to be made in a particular tissue, you can detect the mRNA used to make it by taking the reverse transcriptase approach. You can even detect the particular cells in which it is being made by means of an in situ histochemical version of the test. There are limitations of course. You need to know at least some of the sequence of the gene, and detecting the mRNA does not prove that the protein is being made.
The polymerase chain reaction allows amplification from a convenient cell source of any desired gene sequence. The understanding of transmissible spongiform encephalopathies has been greatly assisted by studying the prion protein (PrP) gene, which seems to play a central role in the pathological process. The region of the gene particularly concerned with susceptibility to the disease has been identified, and this can be amplified and sequenced. For example, the result may show that a particular person has an amino acid substitution (deduced from the nucleotide sequence) that makes them susceptible to the iatrogenic form of Creutzfeldt-Jakob disease or to the familial type.3
In other contexts tissue typing may now be performed by probing the cell nucleic acid rather than using serological methods, and the tiny amounts of tissue obtained by chorionic biopsy can be probed for the presence of abnormal genes in the very early fetus. Foreign genetic material–of viruses or bacteria, for example–can be detected with great sensitivity and more rapidly than by conventional techniques. Furthermore, virus nucleic acid may be found when no virus can be recovered, perhaps because the virus has been neutralised or because it is present in a form that will not grow in the laboratory. For example, a recent paper suggested that coxsackievirus nucleic acid was present in the blood of patients suffering from insulin dependent diabetes.4
Such findings need to be studied critically from the technical point of view. For example, because the polymerase chain reaction is so sensitive it is possible to contaminate specimens in the laboratory and give rise to false positive results, so suitable control tests have to be run in parallel with the main assay. Of course, only if the whole organism can be grown in the laboratory can its biological properties be established with certainty. However, when the relation between a gene sequence and, say, drug sensitivity is understood– as in the case of HIV5–or is being discovered–as in the case of the tubercle bacillus6 7–tests for the presence of changes in key sequences may give valuable information for assessing and managing cases. There are a few instances–such as Kaposi's sarcoma8 –in which the apparent causative agent can be detected only by techniques based on the polymerase chain reaction, and others are likely to follow.
Not long ago immunoassays and enzyme linked immunosorbent assays (ELISAs) were regarded as tools for research and nothing more. After much development, often in commercial laboratories, kits are now available so that these powerful techniques are used routinely in hospital laboratories for the care of patients. The same will happen with the polymerase chain reaction. But it is important that we review carefully whether using one of these newly refined tests is the best way to obtain the information we need.