primer for bisulfite sequencing in a known unmethylated region - is it safe? - (Mar/17/2010 )
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
That should be fine to me if you have already mapped that site in that cell line. If you are not comfortable about that, you can easily design degenerative primer on that site.
degenerative primers are a good solution...
Thank you pcrman!