U3 AOS1 Topic 9: Polymerase Chain Reaction

Polymerase chain reaction is a technique used to amplify the DNA sample by making identical copies. The amount of DNA is doubled after each cycle of this reaction.

PCR involves the following ingredients:

1.     DNA sample

2.     Taq polymerase enzyme

3.     Nucleotide bases

4.     Sequence specific DNA primers(forward and reverse primers)

PCR is carried out in a thermal cycler which is an apparatus that can change the temperature for temperature sensitive reactions. Following are the steps of PCR:

1.     Denaturation: DNA sample is denatured by heating at 90-95°C. Heat breaks the hydrogen bonds between the nucleotide bases of DNA strands so that strands are separated to form single stranded DNA strands.

2.     Annealing: The temperature is cooled to 50-55°C. Cooling allows the primers to bind (anneal) to their complementary sequence on single stranded DNA. Forward primer binds to the template strand (non-coding strand) at 3’ end whereas, reverse primer binds at the opposite strand called as coding strand of DNA at its 3’ end.

3.     Elongation: The DNA strands are heated again to 72°C which allows the Taq polymerase enzyme to synthesize complementary strand of DNA. Taq polymerase works at optimum temperature of 72°C. It binds to the primer and initiates elongation to make double stranded DNA.

4.     Repeat: The cycle completes after the three steps. The cycle is repeated to make more copies of DNA.

Importance of PCR:

When the DNA sample is insufficient, the scientists carry out PCR to make many identical copies of that DNA sample to do further analysis on DNA as:

·        Forensic testing

·        Paternity testing

·        Analyzing fragment of genes for genetic diseases