Polymerase Chain Reaction (PCR) is a widely used technique in biotechnology and molecular biology to amplify the sequence of genetic material. This amplification help in study that genetic sequence easily without losing the original copy of genetic material.
During PCR we simply makes billions to trillions of copies of particular genetic sequence. As genetic material is very small in size and a sequence of it will also delicate to Handel under complex experiments. Making numerous copies of genetic material help them handling properly while doing experiments with genetic material or doing sequencing of genetic material.
Polymerase Chain Reaction was first conducted by Kary Mullis in the 1980s. It is based on the concept of making copies of genetic material complementary to the parent strands.
PCR is based on using the ability of polymerase to synthesis DNA complimentary to the parent DNA strand. Because DNA polymerase can add new nucleotides to pre-existing 3’-OH, it needs a primer from where it can start. This makes researchers to amplify only a sequence from DNA. At the end of PCR the specific sequence will be accumulates in billions to trillions copies.
Components of PCR
DNA template: it is the sample DNA that contain the target DNA sequence. As we know DNA is a double helix, to do replication process it’s double helix must be unfolded so that enzyme DNA polymerase can act on it and can amplify it. This unfolding can be done by applying high temperature to the DNA sequence artificially. This will unfold DNA strands. Now enzymatic reaction can be started to amplify the DNA sequence.
DNA Polymerase: it is a type of enzyme that synthesize new DNA strand complementary to the original DNA strand. Now because both DNA strands are complimentary to each other DNA Polymerase can start from either to amplify DNA sequence.
The first and most commonly used of these enzymes is TaqDNA polymerase (fromThermis aquaticus), where as PfuDNA polymerase (fromPyrococcus furiosus) is used widely because of its higher fidelity when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant.
Primers– short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer. Polymerase start synthesize new strand of DNA at the end of primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates)- single units of the bases A, T, G, and C, which are essentially “building blocks” for new DNA strands. RT-PCR(Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with enzyme reverse transcriptase.
Limitations of PCR And RT-PCR
Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a “plateau effect” occurs, making the end point quantification of PCR products unreliable.