BSP - favors amplification of unmethylated template? - (Dec/14/2005 )

Hi,

I always get good answeres from you, so here is a new question about bisulfite sequencing...
If you have tumor cells (say methylated) and also contamination of normal cells that are unmethylated - can the unmethylated template be favored in the PCR? With less GC the unmethylated sequence would be easier to amplify and after a lot of cycles perhaps you don't see the methylated template since it is not amplifyed as the unmethylated. Is this something that I need to worry about? Is there some way to test it?

Thanks in advance.

hi molbio,

strictly speaking if you are talking about bisulfite PCR and seqeuncing, I would have to say there is no evidence in the literature to say there is inherent PCR bias. This can be seen where people have bisulfite sequenced an imprinted DMR and shown approximately 50% or clones are methylated and 50% are not so this would suggest the absence of bias, however, this is what is presented in the papers and actually seen experimentally could have differed but I don't know.

MSP is a very sensitive technique that would pick up both methylated and unmethylated templates, there is now a technique called headloop PCR and exploits this potential bias to the extreme.

I once read from a paper that bisulfite sequencing PCR favors unmethylated DNA. I could remember what the reference is and whether the authors have verified this or just a speculation by the authors.