One 'T' missing on one allele after bisulfite treatment - (Aug/04/2005 )
Dear Nick and pcrman,
I am puzzled by my BSP result...I found there is one 'T' missing on one allele after the bisulfite treatment. This leads to an outframe deletion at the 5'UTR of my gene. genomic DNA sequencing did not show this phenomenon. Have you ever met this problem? or will this be significant in my gene since I suspect it is imprinted but no literature reports before?
I guess the "T" was a "C" before bisulfite treatment, right? Are you doing direct sequencing or cloning? Is the "T" on the primer? In how many samples have observed this missing "T"?
This could be just a sequencing error, I don't think bisulfite treatment will remove the cytosine from DNA sequence.
Thanks for your reply pcrman!
I am also wondering how the bisulfite treatment can remove a cytosine?! Yes, the "T" was a "C" before bisulfite treatment, and it is not on the primer. I checked with both forward and reverse primers, totally I have checked 4 individuals, 3 patients 1 normal control, they are the same performance. I did the direct sequencing, now am waiting for the cloning results...
Hi, PCRman. the cloning sequencing shows there are 1, 2, 3 cytosines disappear respectively after the methylation...May I ask if you how to explain it?
do you mean that the cytosines have changed to another base, such as a T??
or that base position is missing completely?
The base position is missing completely. Before treatment, the sequence is CCCCCTCCCTCT(12nucleotides), after bisulfite treatment, there are 4 different sequences:TTTTTTTTTT(10), TTTTTTTTTTT(11), TTTTTTTTTTTTT(13) and the normail 12Ts one. This is the cloning PCR results, the direct sequencing shows unorderly deletion...)
Does anyone have any idea about this? Thank you very much.
I think I know your problem
PCR polymerase slippage, with such a long run of T's there are bound to be errors from the polymerase, especially if you are using Taq. The clones you have isolated have picked up templates with polymerase slippage error. I would try using a more processive polymerase and the problem will go away.
Thanks Nick! I will try another high fidelity polymerase.
I would try the Finnzyme/NEB Phusion enzyme. Also, I would use a lower than normal extension temperature, given the very low GC content being amplified.