Is there a PCR length limitation after Bisulfite treatment? - (Nov/29/2004 )

Hey,

I am analyzing the methylation status of two regions: one ecompasses 500bp, the other is around 800bp. Could I just designed primers at flanking sites and amply these two regions? I found usually only 200-300bp were used to amplification for Bisulfite sequencing in most referecne. Is there any PCR length limitation for this method.

Thanks a lot ,

Jimmy

I have not tried PCR size bigger than 450 bp but have seen in one paper that over 1 kb was amplified from bisulfite modified DNA. I doubt about it.

I follow the advice of most authors that 300 bp is about as large as one can go because bisulfite modified DNA is very fragile. You may want to do a nested PCR to increase product-- your first product might be 500 or 800 bp but you might not see a visible band. Also it's ok to just know the status of 300 bp or so if you are seequencing a CpG island-- it will give you a taste for what you are looking at and you can amplify the rest of the region separately if you think you have something significant.