Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, is a scientific technique used to amplify a specific DNA sequence to produce millions of copies. PCR technique is used in many molecular laboratories because of its simplicity as it only takes hours to amplify the DNA sequence of interest. The PCR reaction takes place in a thermo-cycler, which is programmable heating block which cycles between denaturing, annealing and polymerization temperatures.
Typical PCR amplification reaction includes the following key elements:
1) DNA template containing DNA region that is to be amplified.
2) Thermo-stable DNA polymerase with high temperature optimum ~ (70°C).
3) Two primers, which are typically about 20 nucleotides long and are complementary in sequence to the ends of the target DNA.
4) Deoxynucleotide triphosphates (dNTPs), thermo-stable DNA polymerase use these building blocks (dNTPs) to synthesize a new DNA strand complementary to the target sequence.
5) Reaction buffer solution, which provides an appropriate chemical environment for the optimum activity and stability of the DNA polymerase.
1) Denaturing Step:
PCR chain reaction starts with a denaturing step. Samples are heated to 94– 96 degrees Celsius. The heat denatures the DNA by breaking the hydrogen bonds between the two DNA strands. The result of this step is single-stranded DNA molecules.
2) Annealing Step:
After the two strands of the DNA are separated, the temperature is lowered to 50-60 degrees Celsius. During this step, the primers anneal (form hydrogen bonds) with their complementary sequences in the target DNA. The primers and the target DNA follow the base pairing rule: Adenine (A) base pairs with thymine (T), and cytosine (C) base pairs with guanine (G).
3) Extension/Elongation Step:
After the primers are annealed to the target DNA, the temperature is raised to 72 degrees Celsius. The thermo-stable DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding Deoxynucleotide triphosphates (dNTPs) that are complementary to the template strand in 5' to 3' direction.
One cycle of denaturing, annealing, and extension leads to two double-stranded copies of the target DNA. The second cycle leads to four copies of double-stranded target DNA, the third cycle results in eight copies double-stranded target DNA, and so on. Therefore; with each cycle, the number of copies of the target sequence doubles and by the end of 25 cycles there are 33 million copies of the target DNA sequence. As PCR only takes few hours to make numerous copies of target DNA sequence, it is widely used in research laboratories and genome projects.