How to tell incomplete bisulfite treament from real methylation - (Oct/03/2005 )

How can tell the the incomplete bisulfite treament from the real methylation? Because in my bisulfit sequence PCR, I find some likely non-CpG methylation sites, like CTG, CTT and CTA? ALso, some CG are methylated.

hi mou1,

what genomic DNA are you working with? Plant? Mammalian? Bacterial?

Primary DNA methylation mark in mammalians is CG, however in plants and in bacteria there exists other forms of DNA methylation which could help explain your results.

If not, if you were to perform the treatment again if the methylation is real, you will see it again.

Within your sequence is there are particular block of supposed methylation, ie: are you seeing this form of methylation across 50-200bp stretches and all the C's seem to be methylated? if so, this could also be indicative of incomplete bisulfite conversion due to dirty DNA, ie: protein masking DNA from conversion, or it's a repetitve element that self hybrises in the conversion reaction.

Typically to rid of this problem, I modify the denaturation steps in the bisulfite reaction from 37C to 100C for five minutes to ensure denaturation.

Hope this helps

Nick