PCR Flashcards
1
Q
What does PCR do?
A
Amplify a sample of DNA.
2
Q
What is required for PCR? List 4 components.
A
- Primers
- dNTPs (nucleotides)
- Template ssDNA
- Taq polymerase
3
Q
Why is Taq polymerase used?
A
It is thermostable and high temperatures are needed.
4
Q
What happens to the amount of target ssDNA with each round of PCR (theoretically)?
A
It doubles.
5
Q
What are the 3 main steps in a PCR cycle?
A
- Denature the DNA by heating to 95 degrees.
- Cool the mixture to between 45-72 degrees to allow annealing of the primers.
- Heat the mixture to 72 degrees to allow the polymerase to work.
6
Q
Which region does PCR selectively amplify?
A
The region between the primers.
7
Q
What happens to the DNA fragments over successive rounds?
A
They become closer in length to the distance between the 2 primers.
8
Q
What are the 4 main sources of error in PCR?
A
- Contamination - it is a v. sensitive process, contaminants are amplified along with the DNA.
- Innappropriate priming - primers that are not 100% accurate can still bind, especially if the 3’ end of the primer matches the DNA.
- Taq polymerase is highly error prone.
- GC rich areas of DNA are hard to amplify.
9
Q
PCR can be used to introduce ‘site-directed mutagenesis’. What does this mean?
A
It can introduce specific changes into the DNA sequence for genetic engineering.
