The Polymerase Chain Reaction (PCR) is a technique used in molecular biology to amplify single or few copies of a piece of DNA so that it results in generating millions of copies of a certain DNA sequence. This has become one of the most widely used techniques used in the fields of medicine and molecular biology as it is quick, inexpensive and simple. This was developed in 1983 by Kary Mullis.
For the initiation of the PCR there need to have several components like DNA polymerases, restriction enzymes, a DNA template, two primers/synthetic oligonucleotides or short DNA sequences. Here the template contains the DNA region to be amplified. Two primers used are complimentary to the 3’ end of the sense and anti-sense strands of the template. The DNA polymerase used here is known as Taq polymerase where it is extracted from Thermus aquaticus; a bacterium that live in hot springs because it won’t get denatured with the changing temperature in this technique. Deoxynucleoside triphosphates (dNTPs), the building-blocks from which the DNA polymerase synthesizes a new DNA strand are also added.
The reaction initiates with the heating of the reaction mixture to 95-97 °C. So then comes the denaturation step. With the increase in the temperature it denature DNA into single strands by demolishing the hydrogen bonds between the complimentary bases. Then the temperature is dropped to 50-65 °C and that allows primers to get attached to the single stranded DNA strands (template). Stable hydrogen bonds are formed between the template and the primer because primers complement to the nucleotide sequence of the 3’ end of the two template strands are used. This is named as annealing. The next step is the extension and that starts over with the binding of DNA polymerase to the template-primer hybrid. The temperature at this stage depends on the type of the polymerase used in the reaction. Taq polymerase has its optimum activity around 75-80 °C and normally temperature of 72 °C is used.
The DNA polymerase synthesizes a new complementary strand against each template strands by adding dNTPs in 5’ to 3’ direction. The duration for this step rely on the polymerase enzyme used and the length of the fragment to be amplified. Then again temperature is dropped to 70-74 °C at the elongation step to make sure that all the single strands have their complementary strand. And this cycle continues and you get a reasonable copies of the target nucleotide sequence.
The application of PCR technology is very wide. It is used mainly in genetic engineering, forensics, cloning and sequencing the genetic material and in medical research. It can be used to diagnose diseases like AIDS, middle ear infection, tuberculosis and Lyme disease. This can also be used to detect the presence and quantity of known genetically modified organisms by detecting the known modified DNA sequences.
When this technique is used for detecting a specific DNA segment it’s known as qualitative PCR. This is a sensitive method and it enables to identify DNA in a simple solution. This qualitative PCR technology is used to identify pathological changes, in forensic medicine to identify individuals, to determine if donated blood is contaminated.
Apart from the qualitative PCR, the quantitative PCR (qPCR) provide more info beyond identification of DNA. It detects how much of it is there. This is applied in medical diagnostic testing, in basic researches. This quantitative technique is used in search of targets i.e. the sites in the body where new drugs can have an effect on and thus it relates to the finding of new genes or observed variances.
In these qPCR (real-time PCR) methods apart from the components we use for the normal PCR reaction it needs a reporter that bind to the products formed and reports its presence. Fluorescence is used as the detection method and dyes such as SYBER Green I and BEBO is used widely. It is normally taken in to account as the quantity of double stranded DNA amplified is directly proportional to the amount of fluorescence.
Like any other technique this also does have some limitations. When there are inhibitors of the polymerase reaction found in the sample, reagent limitation and self-annealing of the product the PCR may eventually stops. So the real-time quantitative PCR method comes into to the role for the detection of amplified amount.