What is the role of PCR in molecular diagnostics?

PCR is basically a DNA (or RNA) copier. It prints a billion copies of a gene (or any DNA sequence) in about an hour. If you are looking for a needle in a haystack, make a giant stack of needles that dwarfs the haystack. That’s what PCR does. You can easily see why it is a popular technique to find rare things like a virus or a single mutation in your genome.

PCR has been the single most revolutionary tool in diagnostics, in probably the last century. The COVID pandemic was managed solely due to PCR, and its variation qPCR or quantitative PCR. Quantitative PCR follows the PCR reaction in real time -- both quantity and makeup of DNA can affect how fast the PCR reaction reaches saturation (or "completion"). Following bacterial food poisoning episodes is facilitated by PCR -- not only finding small amounts of bacteria, but it's specific type and following its spread and where it originated, and whether two episodes of poisoning in different locations were from the same bacterial species, indeed, the exact same strain.

A critical component of PCR is the DNA polymerase enzyme, typically Taq polymerase, derived from the bacterium Thermus aquaticus which is heat-stable, meaning it doesn't denature at high temps used in PCR, making it ideal for this process Denaturation (95°C): This high temperature causes the hydrogen bonds between the complementary DNA strands to break, resulting in the separation of the two strands into single strand Annealing (50-60°C): The exact temperature for annealing depends on the length and composition of the primers but typically ranges between 50 and 60°C. Extension (72°C): The polymerase extends the primers, adding nucleotides (building blocks of DNA) one by one to build a new DNA strand complementary to the template strand.

There is a special type of PCR that is isothermal so you don’t need to run a thermal cycler. It runs at a single temperature and uses a simpler protocol. The result is read quickly and can be done in a doctor’s office (POC or point of care). I think most of the world learned the phrase “PCR” during the Covid era. It has been around for 30 years but was only known by people who use it or pay attention to Nobel prize winning technology.

Yes, contamination is the biggest confounder in PCR, especially trying to find minute quantities of DNA. Scientific cases are known where what was thought to be the presence of a particular DNA sequence in minute samples, was in fact DNA from the air in the lab that had been using a similar sample in the same space for a while. Though this can be mitigated, running proper negative controls is crucial due to very high sensitivity of PCR>

While PCR is a powerful tool, it requires careful design of primers, control of contamination, and accurate temperature cycling. PCR can also sometimes amplify non-target DNA sequences, leading to false positives. Advances in PCR include qPCR, which allows for the quantification of the DNA. This is particularly useful in diagnostic tests, where the amount of pathogen DNA present can be indicative of infection severity. PCR has numerous applications, including DNA cloning for sequencing, gene cloning and manipulation, gene mutation analysis, paternity testing, detection of bacterial and viral infections, and diagnostic testing for genetic diseases.

Any good scientist who does a well designed experiment or process uses proper controls to be sure they get the right answer every time. This is supremely important for a diagnostic test. Patients lives may depend on it. PCR is really good at finding contaminants and making a billion copies of them. This gives you the wrong answer. Most labs have pre-PCR and post-PCR areas to avoid cross contamination. Post-PCR you have a billion copies of something and if 1 copy gets into a new PCR, you will get another billion copies of it. It is easy to contaminate a reaction and not know it - giving you the wrong answer. Special pipettes are used to carefully move liquid around because 1 copy could escape that way. Good technique is required.