In this article we will discuss about the Polymerase Chain Reaction:- 1. Steps of Polymerase Chain Reaction 2. Advantages of Polymerase Chain Reaction 3. Applications.
Steps of Polymerase Chain Reaction:
PCR uses DNA polymerase to amplify repetitively targeted portions of DNA. The amplified DNA sequence can then be analysed by southern hybridization.
i. Primer Construction:
(a) It is essential to know the nucleotide sequence of short segments on each side of the target DNA.
(b) The nucleotide sequences of the flanking regions are used to construct two single- stranded oligonucleotides, usually 20 to 35 nucleotides long.
(c) These synthetic oligonucleotides function as primer in the PCR.
ii. Denature the DNA:
The DNA to be amplified is heated to separate the double-stranded target DNA into single strands.
iii. Annealing of Primers to Single-stranded DNA:
The separated strands are cooled and allowed to anneal to the two primers.
iv. Chain Extension:
(a) DNA polymerase and deoxyribonucleoside triphosphates are added to the mixture to initiate the synthesis of two new chains complementary to the original DNA chains.
(b) DNA polymerase adds nucleotides to the 3′- hydroxyl end of the primer, and strand growth extends.
(c) After one cycle of replication, the reaction mixture is heated again to denature the DNA strands.
(d) Each DNA strand binds a complementary primer and the cycle of chain extension is repeated.
(e) Typically 20 to 30 cycles are run during DNA amplification.
(f) Each newly synthesized polynucleotide can act as a template for the successive cycles.
Advantages of Polymerase Chain Reaction:
PCR is so sensitive that DNA sequences present in an individual cell can be amplified. The isolation and amplification of a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques.
Applications of Polymerase Chain Reaction:
i. The synthesis of the mutant DNA is allowed by PCR in sufficient quantities without cloning the altered DNA.
ii. The long latency period of viruses such as HIV is difficult to detect at the early stage of infection by the help of conventional methods. PCR gives a rapid and sensitive method for detecting viral DNA sequences.
iii. The isolation of DNA from a single human hair is quite ample to determine whether the sample comes from a specific individual.