primer for bisulfite sequencing in a known unmethylated region - is it safe?
Posted 17 March 2010 - 02:24 PM
I have a question that I still couldn`t solve in any publication or forum.
If I`m mapping the methylation status of a promoter region in a determined gene in a determined cell line, and I`ve seen that a part is unmethylated, and I want to continue searching downstream, but I`m having difficulties in designing primers `cause this region is really rich in CpGs. Can I design primers annealing in previous seen unmethylated segments to amplificate this next region withou having a bias?
I mean, intuitively i could say that there was no problem, and some labs use to put CpG normally in this primers to overcome bias and etc...
but, since I`m new in doing this, I don`t feel confident enough to this this, this the why of my preoccupation in this case, even knowing that the possible annealing region is unmethylated.
I fear having criticisms in my work If I do this without a basement, so if somebody knows any similar situation in some publication, please let me know, but any suggestion or oppinion will be very helpful
tks a lot
Posted 19 March 2010 - 06:23 PM
Posted 31 March 2010 - 06:21 AM
Thank you pcrman!