Well PCR is one of the most used techniques in molecular biology. It’s an amazing technique which acts like xerox, for the DNA. It amplifies the DNA of interest, in just a small tube.
What is PCR and how is it carried out?
PCR stands for Polymerase Chain Reaction and is carried out in series of cycles. It was developed by a biochemist Kary Mullis in 1983 (Read more about him here). This technique is used to amplify the desired DNA sequence.
In this technique, the given DNA template is first denatured to single strands. The single strands of DNA are then allowed to bind with the primers provided. A primer is complementary to a regions flanking the gene/ DNA Sequence of interest. The DNA polymerase then adds the nucleotide bases and extends the DNA template. The increase in the gene copy number can be visualized using either PAGE or AGE.
As obvious, the major ingredients:
- DNA template: desired gene or sequence of DNA to be amplified.
- Primers: provide 3′-OH end to DNA polymerase. Two types of primers are added, forward primer and the reverse primer.
- Nucleotide bases: dATP, dCTP, dGTP and dTTP.
- DNA polymerase: usually from the bacterium Thermus aquaticus known as Taq polymerase.
- Buffer: to provide the right conditions (pH and ions)
The reaction is carried out in an instrument called Thermocycler which provides cycles of temperatures. There are range of different types of Thermocyclers, depending on the needs and modifications.
Steps in PCR:
1. Denaturation:
The two strands of DNA are held together by the weak hydrogen bonds. At higher temperatures the hydrogen bonds break down and the double stranded DNA denatures to two complementary single strands (see fig 1).
This is what is done in the first step of PCR. The temperature is set to around 94⁰C, to denature the DNA double helix. The temperature varies with each DNA sequence, and depends on the base pairs. Higher temperature is required to separate strands with more C-G than A-T.
2. Annealing:
Once the DNA has been denatured, the temperature is lowered to around 55-65⁰C to allow the primers (reverse primer and forward primer) to anneal to the complementary bases along the gene of interest. The forward primer binds the template (sense) strand and the reverse primer to the complementary (anti-sense) strand (see fig 2).
The primers are designed to bind the 5′ end of the gene of interest and provide the 3′-OH group for the extension by DNA polymerase.
3. Extension:
The temperature is set to around 72⁰C to allow the DNA polymerase to bind and add the nucleotides along the length of DNA and extend the DNA of interest. A special heat stable DNA polymerase called Taq polymerase (from Thermus aquaticus) is used.
As in the replication, after one round of these three steps, one copy of gene gives two copies of the same gene, hence there is an exponential increase in the number of copies of the gene with each cycle.
Normally the cycle is repeated around 20-40 times resulting into synthesis of around billions of copies of the DNA.
Detection:
After all cycles are run, the amplification products of PCR are checked using electrophoresis. The thickness and the position of the band helps to identify the success of the experiment. Darker the band, more the gene copies. With help of DNA markers we can identify if the right gene (base pairs) has been amplified.
In the above figure (fig 4), it can be seen that the 4 different samples have the particular gene present. The genes have been successfully amplified.
Current status:
PCR has been modified and upgraded to give various new versions of PCR, like real time PCR, reverse transcription PCR and many more.
Applications:
Used in various medical diagnostics, DNA cloning procedure, forensic field, sequencing of DNA, DNA fingerprinting (eg. AFLP) and more.
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References for the images:
Thermofisher (also read for more info)
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