The PCR (Polymerase Chain Reaction) is an important breakthrough in molecular biological sciences and won Katy Mullins the Nobel Prize in 1993. PCR is all about amplifying DNA strands with the Taq DNA polymerase (obtained from the thermus auquaticus bacteria in several hot springs) and the Pfu DNA polymerase (a subsequent discovery from the pyrocucus ferocious following its fidelity when complementing DNA).
What is the Polymerase Chain Reaction all about?
The chain reaction involving DNA polymerase and short pieces of single stranded DNA (called primers) to synthesize a new DNA strand complementary to the template strand (called amplicons) is called the PCR or the Polymerase Chain Reaction. The PCR cannot work without a primer because it needs a template strand (rich in the 3′-OH group) to which it can add a nucleotide and create a self-sustaining DNA strand.
What are the components that a Polymerase Chain Reaction requires?
In order to produce successfully amplified DNA strands, a PCR requires the following components:
- A template DNA strand - The PCR needs a template DNA strand to create a complementary strand. The pre=existing DNA template is subjected to extreme temperatures to separate the DNA strands from each other and use them in creating self-sustaining individual DNA strands.
- DNA polymerase - DNA polymerase are heat-resistant enzymes (for example, the Taq DNA or the more popular Pfu DNA polymerase) that are used to amplify and synthesize the individual DNA strands of the template provided such that the resulting sequence is complementary to the existing sequence.
- A primer - Primers are short DNA strands that are complementary to the sequence targeted. During the PCR, the polymerase uses the 3′-OH groups from the primer and adds the nucleotides to create an individual DNA strand.
- Nucleotides - Also referred to as dNTPs and deoxynucleotides triphosphates, nucleotides are the “building blocks” of DNA and contain single units of the bases A, T, G, and C.
How is the Polymerase Chain Reaction carried out?
Experts bring together a DNA template and the DNA polymerase (could be the Taq or the Pfu) and use a primer and dNTPs to begin the DNA polymerization reaction. The primer is added both to the normal 5′ direction of the DNA strand and the opposite direction for proper DNA sequencing. When the DNA template is melted at 95°, the primers stick themselves to the gene strand at 45° (also called annealing) and there are two strands created at 72° by the polymerase. Thus, the DNA quantity doubles in a jiffy.
Similarly, there can be advanced PCR chain reactions that help you create more than 1000 strands of DNA complementary to a template provided within an hour or so.
Since the PCR reaction deals with micro managing DNA strands and amplifying them to create large clones of a template strand, it is relevant to the forensic science industry that deals with individual DNA to discern the cause of death or injury at a cellular level, for instance, the O. J. Trial.
The PCR is also used in areas of cell mutation and hybridisation technology, DNA cloning and sub cloning, DNA sequencing, and discerning genetic disorders. Moreover, the PCR can be used to detect cellular infections in the body and cure diseases before they turn life threatening. The PCR thus, has myriad industry and laboratory applications.
Limitations of the PCR
Although the PCR is an example of path breaking technology, it has several limitations and challenges. Some of them are illustrated below:
- The presence of divalent cations and nucleotides must exactly match the synthesis specification, or a non-specific DNA amplification will take place.
- Primers need to be used with care. If they don’t exactly match the template, there are possibilities of strand mutation. Moreover, specific attention has to be paid to the annealing since the primers tend to (sometimes) anneal to themselves leading to defective DNA synthesis.
- The most crucial challenge while attempting a PCR is hygiene. Toxins, blood drops, and other substances may contaminate the DNA strand and give you misleading results, especially when used in forensic laboratories. Thus, you must make sure the PCR components are used in isolation and there is no other substance taking part in the reaction.
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