4C: The Polymerase Chain Reaction

What is PCR

a DNA manipulation technique that amplifies DNA by making multiple identical copies

What are purposes of PCR

whenever there is an insufficient DNA sample for testing

• Forensic testing samples of bodily fluids

• Paternity testing

• Analysing gene fragments for genetic diseases.

What materials are needed for PCR

• DNA sample

• Taq polymerase

• Nucleotide bases

• DNA primers

What are the stages of PCR and the temps at which they occur

1. Denaturation 90-95C

2. Annealing 50-55C

3. Elongation 72C

Repeat!

Denaturation

DNA is heated to approximately 90–95 °C to break the hydrogen bonds between the bases and separate the strands, forming single-stranded DNA

Annealing

the single-stranded DNA is cooled to approximately 50–55 °C to allow the primers to bind to complementary sequences on the single-stranded DNA.

Elongation

the DNA is heated again to 72 °C, 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

Repeat!

the cycle is repeated multiple times to create more copies of DNA

What are the primers used

forward and reverse primers

Forward primers

binds to the start codon at the 3’ end of the template strand

causes Taq polymerase to synthesis a DNA strand in the same direction that RNA polymerase would

Reverse primers

binds to the stop codon at the 3’ end of the coding strand

causes Taq polymerase to synthesis a DNA strand in the opposite direction that RNA polymerase would