A Powerful Technique: PCR- An Insight
PCR, or Polymerase Chain Reaction is a temperature guided technology comprising of the following basic steps:
Initial denaturation (temperature is based on the GC content of the template DNA),
annealing of primers to the DNA strands,
the elongation step with the help of Taq DNA polymerase.
The PCR process can be represented by the typical 'S' shaped curve.
Variants of PCR and its applications
PCR, a temperature-guided technique developed in the 90’s.
These are several variants of PCR; each variant attends to a very specific requirement.
There is Quantitative PCR which uses fluorescent dyes or fluorophore containing DNA probes to measure the amount of amplified product, as the amplification progresses.This is widely used to measure the specific amount of nucleic acid (DNA or RNA) in a sample. A Multiplex PCR, most commonly used for DNA fingerprinting has several primers attaching to different target gene sequences. For example, while checking gene mutations, 6 or more amplifications may be combined.
Several sequencing and hybridization probing applications require the amplification of only one of the DNA strands. This is carried out by an Asymmetric PCR which preferentially amplifies one of the target strands. When the sequence to be amplified is extremely large, we use a Nested PCR. In the first PCR, we amplify a longer fragment which contains the desired sequence along with some non-target regions. In the successive PCR, we use primers to specifically amplify the target sequence. For direct screening of bacterial colonies, a Colony PCR is used. A small number of cells are directly transferred from each individual colony to the respective PCR mix, and a modification is made in order to make the cells release the DNA; the PCR is started with an extended time at around 95°C. Sometimes, pretreatments and extensions are made in the normal PCR to cater to specific needs which result in RT-PCR, PCR-RFLP, RACE-PCR, etc.
PCR has variegated uses in the field of medical science. Real Time PCR is extensively used for quantification of expression of specific genes. It has been used for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases. (Overbergh et al., 2003). When a comparison was drawn between PCR and Southern Hybridization for routine detection of IgH rearrangements, it was found that PCR was more specific as well as more sensitive. 90% of IgH genes found to be rearranged by Southern hybridization are detected by the PCR technique. Additionally, patients’ samples having clonal IgH gene rearrangements were detectable by PCR alone (Lehman et al., 1995).
This technology (TAIL-PCR) has also been exploited for large scale projects like Chromosome Walking. A (TAIL)-PCR utilizes nested sequence specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficienciesof specific and non-specific products can be thermally controlled. One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment. This sequence is then preferentially and geometrically amplified over non-target ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles (Whittier et al., 1995).
Polymerase Chain Reaction is widely used for the purpose of disease diagnosis as well. For instance, PCR can confirm the presence of certain mutations in the genome which predispose the individual towards certain diseases like Breast Cancer. Presence of Mycobacterium tuberculosis in a person’s blood sample can also be corroborated via PCR.
PCR applications aren’t limited to the medical field only. In a research, Duplex PCR was used to identify bovine and water buffalo DNA from milk used in making mozzarella cheese. DNA concentrations as low as 1 picogram were detected (Stefano et al., 2001).
From research point of view, PCR is used for DNA sequencing, as done in the Sanger’s method involving the use of di-deoxyNTPs. It is also used in medical forensics for genetic fingerprinting and paternity testing.
Limitations of this technique arise from the fact that DNA polymerase is prone to error, which in turn causes mutations in the amplified DNA. Additionally, the specificity of the PCR fragments can also mutate the template DNA due to non-specific binding of primers. Designing the primers is a cumbersome process per se. Despite the limitations, PCR has reached a high level of reliability and practical ease that makes it a well suited technique not only for research purpose but also in a clinical setting.
- FORWARD PRIMER
- REVERSE PRIMER
- Taq DNA POLYMERASE