Polymerase Chain Reaction(PCR)

The polymerase chain reaction (PCR) technique was first developed by “Kary Mullis” in 1985,. By using the PCR technique. We can multiply (or) increases the DNA from microgram (mg) level to the higher amount level. The PCR process doing by the machines, they are “Thermocyclers”.

The PCR is carried out in vitro ( in test tube). It utilizes:

• Desired DNA fragment
• Two nucleotide primers (about 20 bases long)
• The four deoxynucleoside triphosphates E.g.: TTP, dCTP, dATP, dGTP
• Heat stable DNA polymerase E.g.: Taq polymerase (isolated from bacterium the “Thermus aquaticus”), Pfu polymerase (isolated from “Pyrococcus furiosus”)

Procedure:

Before starting PCR, reaction mixture will be prepare. It contains amplifable DNA, excess of the two primer molecules, four deoxyribonucleoside triphosphates and DNA polymerase.

Step 1:(Denatureation step): The reaction mixture is heated to a temperature (usually 90 to 98⁰C) that assures DNA denaturation.

Step 2:(Annealing step): The mixture is now cooled at a temparature (40 to 60⁰C) that permits annealing of the primer to the complementary sequences in the DNA. These sequence locate at 3’ ends of two strands of the desired segments

Step3: The temparature adjusts for the primer extending process through the 3’-hydroxyl group of primers (only annealed primer molecules). The primers are extended towards each other so that the DNA segment lying between the two primers is copied . In this polymerization “Taq polymerase” enzyme plays an important role in the polymerization process in the replication in vitro. The completion of the step3 completes the first cycle of amplification each cyle may take place one to three minutes.

Step 4: The next cycle of amplification is initiated by denaturation, which seperates the DNA strand from newly synthesized complementary DNA strand.

Step 5: Annealing allows the primers to base pairs with both the new and old strands, the totla number of strands being twice their oiginal number.

Step 6: Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of step1. This completes the second cycle.

Thus at each cycle, both new and old strands anneal to the primers and serve as emplates for DNA synthesis. The end of ‘n’ cycles 2ⁿ copies of the segment are expected. The cycle may be repeated upto 60 times, but usually 20 to 30 cycles are adequate.

After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and can be used for the desired purpose.