What is the advantage of nest PCR in methylation analysis - (Feb/07/2006 )
I am designing primer for methylation analysis. I read several papers and found nested PCR was applied. I have several questions about this. 1) primer design. When designing the first primer set amplifying a longer DNA fragment (see 400bp), I should use the bisulfite-modified sequence, right? 2) The optimal product size for MSP is 200bp.If so, is it ok to amplify 400bp PCR product by using bisulfite-modified sequence. 3) Is there big advantage to perform nested PCR for methylation analysis?
By the way, it is the first time for me to design MSP from mouse genes. Althoug the primer design program output several sets primers for methylation analysis, no CpG island were identified in most genes I selected. It seems like CpG island in mouse gene is not so rich as that in corresponding human genes. Will these primers give good result?
It will be very appreciated if you can give me some guide or share your experience with me. Thanks!
yes pick primers to your bisulfite converted sequence, the reason for performing two rounds is that your starting converted DNA is very little but can be recovered by two rounds or PCR amplifications.
CpG islands are a good starting point for methylation analysis however it is becoming apparent that differential methylation can occur at sites that are not defined by a CpG island, and you should check which definition your program is using, Takai's definition >500bp, GC>55%, obs/exp>0.65 is more stringent than the classical definition of >200bp, GC>50%, obs/exp>0.6.