Why proofreading Taq can't be used in MSP - (Jun/03/2005 )

Hi

I just learnt that proofreading polymerase (i.e polymerase with 3'-5' mismatch repair) can't be used in MSP, do anybody know why? And what problem will it cause? I have tried to ask the tech support from Chemicon, but they don't know neither!
Thanks for any help.

I'd assue it is because proof-reading polymerases can't recognise methylated DNA, but i don't know for sure.

moz is on the right line. However it is because you want to detect differences in the DNA that tells you if it's methylated or not.

Because you are amplifying from a heterogeneous population of bisulfite converted DNA, the proof reading enzyme will induce an unwanted bias towards one population or another giving rise to possible artefacts from the reaction.

Ideally a Taq with no proofreading ability should be used as the templates are amplified as is.

Nick

Hi Keentolearn,

Where did you get the source that proof reading taq can't be used for MSP?

hi mrpcr,

that comment pertains more towards bisulfite PCR and sequencing. However, with msp, you are relying on the primer's ability to discriminate a C from a T mismatch say, and the proof reading enzyme could correct for this mismatch giving rise to a false positive or a false negative in the reaction.

Hope this clears it up a bit more.

Nick

Hi mrpcr

Sorry for the late reply. This says in Chemicon CpG modification kit.