Polymerase chain reaction (PCR) is a revolutionary laboratory technique that enables the replication of a specific DNA sequence. Using PCR, millions of copies of a target DNA can be easily synthesized within a short period of time.
It is a relatively low cost and sensitive technique that has paved the way for ground-breaking genetic research and diagnostics over the years. Since its advent in the 1980s, this technique has found a variety of applications in innumerable scientific disciplines, thanks to its specificity and versatility.
Components of PCR
The sample DNA containing the target sequence.
The enzyme that synthesizes fresh DNA strands complementary to the target DNA sequence. Taq DNA polymerase is the most commonly used polymerase in PCR. Taq refers to Thermus aquaticus, a thermophilic microorganism from which this heat-resistant DNA polymerase is extracted.
Taq polymerase can be stable at temperatures as high as 95oC, making it ideal for PCR. Another commonly used organism is the Pfu DNA polymerase, found in the heat stable organism Pyrococcus furiosus. Both the enzymes are capable of generating new DNA strands using primers and a DNA template.
Tiny segments of single-stranded DNA that help DNA polymerase to initiate the synthesis of new DNA strands. Primers are complementary to the target DNA sequence.
Individual bases A, T, G, and C, which are the building blocks of DNA. They act as raw material for new DNA synthesis.
Buffer (usually MgCl2)
A salt solution used to stabilize the reaction components, especially the DNA, and maintain an optimal pH during the reaction.
Provides the liquid environment needed for the chain reaction.
Key Steps in PCR
A high temperature of 95°C is applied to the original double stranded DNA. Heat denatures the DNA by breaking down its weak hydrogen bonds. Thus, the double stranded DNA separates giving rise to single stranded DNA.
The denatured DNA is cooled down to 45 - 72°C allowing the primers to bind to their complementary sequence in the single stranded target DNA. Annealing temperature is a crucial factor as the hybridization of DNA is a temperature-dependent process. If this temperature is too high primer-template pairing will not happen, thus hindering the chain reaction.
Extension of Primer
The mixture is then heated to 72°C, at which DNA polymerase starts to add nucleotides to the primer using the target DNA as a template and extends the primer resulting in new DNA formation.
Repeat Step 1
The above 3 steps are repeated about 30 times for amplification of the newly formed DNA. For each new cycle, the DNA formed in the previous cycles also serves as a template and therefore the amplification is exponential.
Types of PCR
There have been several adaptations of the standard PCR over time. Some of the key variants of PCR are mentioned below:
Reverse Transcription PCR (RT-PCR)
Allows the production of DNA templates corresponding to an RNA sample with the help of an enzyme called reverse transcriptase.
Real Time PCR
An advanced variant of PCR where the entire chain reaction is monitored and data is gathered in real time.
Allows simultaneous amplification of numerous DNA samples
Used to increase the specificity of the amplified DNA and reduce non-specific binding of primers.
Allows amplification of only one strand of the original double stranded DNA
In situ PCR
PCR carried out in the cell or a tissue fixed to a slide