2 replies to this topic

[SimonT]

Hi!

I red on this forum that polymerase for bisulfite PCR shuld be non proofreading. I want to ask is it true and if it is, why?

Which polymerase do you recommend?

Regards!

Simon

[methylnick]

Bisulphite conversion changes all unmethylated C's to U's. And therefore generate non-complementary strands

I believe the proof reading polymerase would either stall or try to correct the mismatches arising from this base change in the first few cycles. This could potentially overestimate or underestimate the methylation calls as you are trying to detect a C or T for DNA methylation.

Nick

[phage434]

The PCR enzyme is only copying a single strand -- the strands are denatured to single stranded.  The problem with proof reading enzymes is the uracil, which poisons the proof reading enzyme copying reaction by stalling the enyzme.