PCR Flashcards
What is PCR?
It’s use?
The polymerase chain reaction (PCR) can be used to select a fragment of DNA (containing the gene of section of DNA youre interested in) and amplify it to produce millions of copies in just a few hours
PCR has several stages and is repeated over and over to make lots of copies
Describe stage 1 of PCR
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and polymerase
Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
DNA polymerase is an enzyme that creates new DNA strands
Step 2 of PCR
The DNA mixture is heated to 90-95°C (for 30 seconds) to denature DNA by breaking the hydrogen bonds between the two strands of DNA
DNA polymerase doesn’t denature at this high temperature, this is important as it means many cycles of PCR can be carried out without having to use new enzymes each time
The mixture is the cooled to 55-60°C so that the primers can bind (anneal) to the ends of the strands
Step 3 of PCR
The reaction mixture is heated to 72-75°C for at least 1 min, this is the optimum temp for DNA polymerase to work.
The DNA polymerase lines up free DNA nucleotides alongside each template strands, adding bases to the primer.
Complimentary base pairing means new complementary strands are formed
Step 4 of PCR
Two new copies of the fragment of DNA are formed and one cycle of PCR is complete
The cycle then starts again - and this time all 4 strands (2 original and 2 new) are used as templates
(2^n , n= number of times PCR was carried out, to work out how many DNA strands produced)
Why is Taq polymerase used in PCR?
The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria found in hot springs.
It can withstand high temperatures (it is a types of DNA polymerase)
