Which polymerase for bisulfite sequencing PCR - (Dec/09/2005 )
Dear PCRman and MethylNick,
as you probably know there is an enormous amount of information on the internet regarding methylation studies and as a fresh researcher with little experience it is difficult to see the trees in the woods. My colleague and me will be doing a pilot methylation study on a couple of genes using bisulfite sequencing and have a question:
Do we need an RT polymerase after the bisulfite treatment of the DNA in order for the U's to be transcribed to A's or will a normal (jump start red?) Taq do?
You don't need a reverse transcriptase to convert 'U's to 'T' because any DNA polymerase can do it. Bisulfite sequencing PCR (BSP) is basically a regular PCR except more cycles are needed.
For BSP, yes, JumpStart red taq from Sigma gives much better results than regular taq.
I second Pcrman,
However, as a cost saving measure, we use conventional PCR master mix (promega) with a manual hotstart (wait for the cycler to get to denaturation temperature then put the tubes in). If you are cost saving, conventional taq will work, as with our situation.
As pcrman said, more cycles are needed, usually two rounds of PCR at 30 cycles each using nested or semi nested primer pairs (for bisulfite sequencing and PCR).
good luck, it's great fun jumping into tthe field, speaking from experience.
Wonderful, thanks a lot.