can we quantify bisufite treated DNA - (Jul/14/2005 )
Since Unmethyalted C--->U, the modified DNA is no long regular DNA or RNA. Can We still use stardard method to quantify it?
indeed you could, but will a spectrophotometer be sensitive enough to detect it? probably not!
Thanks! Methylnick! But I still have two question:
1.If we want to do real time PCR to detect the differece between copy number of methylation allele from the unmethylation allele from different pathogical tissue, how can we do this without a standard curve? If we need the standard curve, we have to quantify modified DNA
2.My boss said the standard DNA concentration value of 1 OD is no longer applicable because the C----->U, so how can we quantify modified DNA?
for your first question, you can construct a standard curve by mixing known ratios of your template unmethylated and artificially methylated (with SssI methylase) and bisulfite treating these samples to construct a standard curve.
ie: depending on your ratios you will get varying Ct values and from this standard you can estimate allelic proportions of your sample DNA.
Indeed your superviosr is correct in saying that OD will not be accurate because C's have converted to U's. Like I said before you won't have enough DNA post bisulfite conversion, to be able to quantify it through OD anyway!