PCR Flashcards
What materials does PCR require?
- a DNA sample
- Taq polymerase
- nucleotide bases
- sequence specific DNA primers
- buffer solution (maintains pH)
What are the processes of PCR?
- Denaturation
- Annealing
- Elongation
- Repeat
What does PCR stand for?
Polymerase chain reaction
What happens in the denaturation stage?
DNA is heated to approximately 90-95 degrees to break the hydrogen bonds between the bases and separate the strands, forming single stranded DNA
What happens in the annealing stage?
the single stranded DNA is cooled to approximately 50-55 degrees to allow the primers to bind to complementary sequences on the single stranded DNA
What happens in the elongation stage?
the DNA is heated again to 72 degrees 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
What happens in the repeat stage?
the cycle repeats multiple times (at least 30) to create more copies of DNA
What is Taq polymerase?
What does the DNA sample do in PCR?
gets denatured and amplified
What do the nucleotide bases do in PCR?
must be constantly available for Taq polymerase to create a new strand that is complementary to the single stranded DNA
What does the Taq polymerase do in PCR?
in the elongation stage it binds complementary nucleotides to the single stranded DNA
What do the primers do in PCR?
join to the 3’ end of single stranded DNA by complementary base pairing to form the first segment of double stranded DNA, allowing Taq to attach and begin extending the DNA strand
What are the two different primers?
forward and reverse
Why are there two different primers needed in PCR?
during denaturation, the double stranded DNA molecule has been separated into two single strands - the template strand and the coding strand
What is the function of the forward primer?
binds to the start codon at the 3’ end of the template strand. This causes Taq polymerase to synthesise a new DNA strand in the same direction that RNA polymerase would function
What is the function of the reverse primer?
binds onto the STOP codon at the 3’ end of the coding strand. This causes Taq polymerase to synthesise a new DNA strand in the reverse direction that RNA polymerase would function
Why is having the two primers necessary?
the 5’ ends of both the template and coding strands are different. As Taq polymerase only functions toward the 3’ end, a primer is needed for both strands to facilitate this directionality
