primers for bisulfite-modified DNA: general questions - (Apr/16/2007 )

Hello everybody,
I am new here and in the "world of methylation". As a beginning, I read most of the 23 pages of the forum… but still have some questions


Background:
I screened for variation in DNA methylation in rat brain following treatment relative to control. From this initial screen, I have several DNA fragments that are potentially differentially-methylated. Now I want to verify these results by bisulfite analysis of the specific fragments. (The same idea as in verifying results of cDNA array study with qPCR)

I want to plan primers for sequencing of bisulfite-modified rat DNA:
My questions are:
1) I read that "The most sequence specific part of your primer is the 3' end" why is that and what is its the practical meaning?

2) After Bisulfite treatment the DNA sequence becomes much less complex and thus I would expect that the primers could recognize many other areas in the genome and produce non specific products.

Do you agree with me? How can I check in advance that the primers that were design for bisulfite modified DNA will not amplify additional areas that are on the rat genome?

3) If I understand correctly, DNA methylation affects gene expression by physically interfering with "machinery" on the promoter. So the functionally-significant methyls are those that are on the antisense strand of the DNA (the strand that is the template for mRNA formation). In other words methylation on the sense strand do not affect gene expression.
SO, If all this is true, I should specifically plan primers for the antisense strand of the DNA, and not choose arbitrary the strand to be amplified. Am I correct?


Thanks in advance for any advice or comment.
Yael

Hi Yael, welcome to the forum!

Thank you for the prompt reply,
Sorry if my question is trivial but I do not understand why the 3' end would have more impact on specificity than the 5' end of the primer?
Regarding the second question (primers that recognize several areas in the genome), does nested PCR can solve this problem, if occurs?

Yael

3' and specifity - oops, I don't know exactly, think it has something ot do with affintiy and thermodynamics - shit, I am just a dump user ...
Nested PCR - it is a question of probability - nested PCR will much less likely show tis problem ... and I would generally suggest it for all bisulfite based analyses.

K.

hi yealcher,

for the 3' specifcity if you can imagine, Taq polymerase extends from a primer by recognising a double stranded part encompassing the primer and the template, with perfect complimentarity Taq will extend from the primer, however, if there are mismatches, within the primer, the primer may not bind to the sequence. With bisulfite and normal primers, the 3' end is most discriminatory because if there are mismatches at this end, it effectively inhibits Taq from extending from the primer because it's not bound to the template.

DNA does become less complex after bisulfite conversion, this is why in my experiments I design primers that are 30mers to ensure sequence specificity. I perform hemi-nested PCR to increase the specificity and this works quite well.

With gene expression, I think DNA is still double stranded and transcription factors and enchancers bind to the sequence which then recruits RNA pol II. Methylation of CpG's affects the structure in that region and recruits repressive proteins to that region that inhibit TF's and enhancers thus shutting down gene transcription. It is only when transcription is initiated when the strands are displaced as the RNA is transcribed.

Welcome to the world of DNA methylation.

Thanks a lot Krümelmonster and methylnick for the time you took to answer me. You really clarified these subjects for me.
Yael

@Nick - thanks for the information on 3' specifity ... reading it, it really seems something one could easily imagine

Krümel