How Does The PCR Test Work and When Can It Be Used?

By Natasha Quinton-Hibberd - Biology Student @ St Catharine's College, Oxford

 

The polymerase chain reaction amplifies DNA in vitro (performed outside of a living organism, for example in a test tube or culture dish). The process occurs in three main steps: DNA strand separation, the annealing of primers and DNA synthesis. During DNA strand separation, the double stranded DNA is heated to 95 degrees Celsius. Consequently, the hydrogen bonds between the base pairs of the parent molecule are broken; the three hydrogen bonds between cytosine and guanine base pairs as well as the two hydrogen bonds between adenine and thymine. This leaves two single strands of DNA that can be used as template strands when the amplification step begins.


The solution is then cooled to 54 degrees Celsius so oligonucleotide primers can anneal. However, this temperature can vary depending on the length and composition of the primer RNA that must bind to the DNA of interest. The composition of the primer RNA is important as it will alter the amount of hydrogen bonds that have to be formed, as will the length of the primer. Cytosine and guanine form one extra hydrogen bond than the other base pair, adenine and thymine. So, depending on the composition of the primer, more or less hydrogen bonds may have to form during the annealing process. The more hydrogen bonds that must form to anneal the primer, the lower the temperature must be for annealing to occur. When the right temperature has been reached, the oligonucleotide primer will anneal.


In the final step of the PCR technique, elongation occurs. The Taq polymerase enzyme, from the organism Thermus aquaticus, works at 72 degrees Celsius as it is a heat stable enzyme. So, this is the temperature for polymerisation of the double stranded DNA molecules. The Taq polymerase elongates from the 5’ to 3’ end. If the PCR cycle is repeated many times, the DNA of interest will be exponentially amplified.


The PCR technique has many applications; for example, medical diagnostics for viral diseases such as HIV. Due to the amplification of DNA that PCR can achieve small amounts of DNA from the human immunodeficiency virus (HIV) can be detected in samples from patients’ blood. As well as this, PCR is a major resource used in forensic science, amplifying the miniscule amounts of DNA that can be collected at crime scenes. This allows these DNA fragments to be further analysed and potentially matched to the perpetrator of the crime through comparing these DNA fragments with DNA collected from suspects.


Recombinant DNA techniques often use the PCR technique. Fragments of DNA containing the gene of interest can be amplified. This is then of critical usage for insertion into the vector. The DNA insert must be of a much higher concentration in the solution than the vector. This will decrease the chance of the vector reclosing without the insert. So, PCR is vital in helping secure the insert of a gene of interest into a vector. The creation of recombinant DNA itself has several varied usages. From the creation of GM crops for agricultural usage, to pharmaceutics and medical uses.


It is clear that the PCR technique is a critical technique in many areas of biology and beyond. Its usage is diverse, from forensic science to medical diagnostics to the mitigation of global issues such as food scarcity through genetic engineering.


Further reading:

  1. Polymerase Chain Reaction. https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/polymerase-chain-reaction-pcr

  2. Applications of the Polymerase Chain Reaction. https://www.bio-rad.com/en-uk/applications-technologies/pcr-polymerase-chain-reaction?ID=LUSNYI15

  3. PCR: Principle and Applications. https://www.intechopen.com/chapters/67558