PCR Flashcards
Between what temperatures is the annealing step of PCR conducted?
50-65°C.
What is the purpose of the initial and final ‘holding’ phases of PCR? What temperatures?
The initial holding phase at 95°C ensures that all dsDNA has become ssDNA.
The final holding phase at 72°C ensures that all DNA products are fully extended.
What is the effect of too much Mg in the PCR mix?
Lower specificity for DNA polymerase.
What do we want primers to have in terms of G and C content?
Primers should have 40-60% GC content and a G or C at the 3’ end.
What are three benefits of having a PCR master mix?
- Reduces pipetting errors
- Minimises contamination
- Faster
How quickly does Pfu DNA pol extend DNA? How and why does this differ from the Taq enzyme?
Pfu DNA pol extends at 1 kb / 2 mins, where Taq can do 1 kb/ min. This is because the Pfu enzyme has a proof-reading element.
A negative control of PCR mix should produce what gel result after electrophoresis?
As there should be no DNA in the master mix, there should be no gel band for the negative control.
What variation of PCR can measure product in real-time, and tell us how much template we began with?
qPCR
What variation of PCR uses RNA as the template?
Reverse transcription (RT) PCR
What is the advantage of droplet digital PCR (ddPCR)?
Rather than a single measurement, the template can be fragmented into thousands of different amplification events.
What variation of PCR uses primers designed to amplify single nucleotide variants (SNVs)?
Allele specific PCR
What variation of PCR uses different primers to test for different strains in the same tube?
Multiplex PCR
What highly variable genomic regions can be used to distinguish individuals?
Short tandem repeats (STRs)
How many times do STRs repeat?
Between 5-50 repeats.
What variation of PCR amplifies longer DNA lengths of 20-30 kb?
Long-range PCR
