Polymerase chain reaction (PCR) is a useful molecular biology technique developed by Kary Mullis in the 1980s. PCR is used to amplify DNA templates into millions of copies of a particular DNA sequence within hours. Due to its powerful ability, PCR is applied in different areas such as; genetic and infectious disease identification, forensics, and research in molecular biology. In contrast to conventional PCR, quantitative reverse transcriptase PCR (qRT-PCR) allows measurement of gene expression quantitatively and in real-time.
In general, a complete PCR reaction requires five basic PCR reagents; DNA/RNA template, DNA polymerase, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs) and PCR buffers.
Selecting the right templates for PCR is vital as different templates are used for the various methods of PCR. For instance, when performing RT-qPCR, RNA templates are required for producing complementary DNA while DNA templates are needed for conventional PCR. It is also important to have DNA or RNA templates in high quality and quantity as this can help optimizing the efficiency of PCR. For this reason, a good PCR template preparation kit is needed for the first step of a successful PCR.
All PCR reactions require a DNA polymerase which can work under high temperature (around 70 °C) as the first phase of PCR involves separation of DNA strands at high temperature (~90 °C). Taq polymerase, which is a heat-stable enzyme isolated from the thermophilic bacterium Thermus aquaticus is a commonly used DNA polymerase for PCR. Some DNA polymerases are engineered to be activated at high temperature only, so as to reduce non-specific amplification at the beginning stage of PCR.
The initiation of DNA synthesis requires primers; short strands of nucleotides (DNA or RNA) which are complementary to the template DNA and serve as a DNA synthesis starting point for the DNA/RNA polymerase. Annealing of primers to single strand DNA requires lower temperature (50-65 °C) than the denaturation step. Once the annealing step is completed, hydrogen bonds will form between the primers and the template DNA.
Deoxynucleotide triphosphates (dNTPs)
Deoxynucleotide triphosphates (dNTPs) are the essential components of PCR as they act as the building blocks of nucleic acids; DNA polymerase cannot synthesize DNA without a supply of dNTPs.
PCR buffers ensure that the PCR reaction is conducted under optimal conditions. The major components of PCR buffer include Tris-HCl, potassium chloride (KCl) and magnesium chloride (MgCl2). Tris-HCl and KCl are responsible for maintaining a stable pH during PCR. Magnesium ions act as cofactors for DNA polymerase so as to ensure proper DNA synthesis function of the polymerase during PCR. PCR buffer is usually available at 10X concentration.
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