Polymerase Chain Reaction (DNA manipulation technique that amplifies DNA by making multiple identical copies)

Materials

• a DNA sample, that subsequently gets denatured and amplified through the polymerase chain reaction

• Taq polymerase, is required in the elongation stage to bind complementary nucleotides to the single-stranded DNA

• Nucleotide bases, for Taq polymerase to create a new strand that is complementary to the single-stranded DNA

• sequence-specific DNA primers, that join to the 3’ end of the single-stranded DNA, allowing Taq polymerase to attach and begin extending the DNA strand

Process of PCR

1. DNA is heated to approx 90-95C to break the hydrogen bonds between the bases to form single-stranded DNA

2. the single-stranded DNA is cooled to approx 50-55C to allow the primers to bind to complementary sequences on the single-stranded DNA

3. the DNA is heated again to 72C which allows Taq polymerase to work optimally

   Taq polymerase binds to the primer which acts as a starting point, and begins synthesising a new complementary strand of DNA

Need for two different primers in PCR

during denaturation, the double-stranded DNA molecule has been separated into two single-strands (template and coding strand)
having both primers is necessary as the 5’ ends of the template and coding strand are different. As Taq polymerase only functions towards to 3’ end, a primer is needed for both strands 

Forward Primer

binds to the start codon at the 3’ end of the template strand, causing Taq polymerase to synthesise a new DNA strand in the same direction that RNA polymerase would function

Reverse Primer

binds to the stop codon at the 3’ end of the coding strand, causing Taq polymerase to synthesise a new DNA strand in the reverse direction that RNA polymerase would function