No modification on reverse strand - (Dec/22/2005 )
I've been lurking for some time but this is my first question.
My first meth experiment worked very nicely. Amplification of p16 bisulfite treated gDNA followed by sequencing. Very nice conversion of all non CpG cytosines - protection of the cytosines in CGs. The problem is that I don't see any conversion in my reverse strand. The G's corresponding to the sense C's are converted to A's but the C's in the antisense strand are not converted. Does this make any sense?
I can't imagine why the C's in the antisense strand are not being converted. Any ideas?
Thanks in advance!
sure does make sense, the nature of bisulfite PCR is that it is strand specific amplification. so if your primers are directed to the "sense" strand all the converted unmethylated C's, become U and by PCR these are converted to T. as the antisense strand is copied, what was a unmethylated C is now a T and therefore the complement of that is an A, hence you get the A. When the antisense strand is copied and comes to a G (from the sense strand) the compliment of this is a C and therefore is incorporated into it.
I think it would make more sense with a diagram, however the nature of the PCR is strand specific, if it were not, it would wreack havoc to the system as I had found before fully knowing the technique.
How do you know the reverse strand is not converted? I am suer you must have used different primers for the reverse strand, right? As Nick has pointed out, after bisulfite treatment, two strands are no longer complementary. How come G's are converted to A's?
Sorry, I'm a loser and never thanked you guys. Thanks!!!
I finally realized that I only had designed primers against the sense strand. Duh....