Bisulfite sequencing primer design query (probably simple) - (Feb/15/2011 )

Hi,

I am new at this - had a look on web and this forum to try and answer this question but failed - suspect it's straightforward. Would like to examine methylation status in FOXP3 and have read a fair number of papers that have recently done this and think I know how to proceed and principals of bisulfite sequencing. In process of setting this up.

What I don't understand is why primers listed in these papers (whether methylation specific or not) can ever include two (or more) G nucleotides in sequence. Whether the primer is forward (and therefore recognises the antisense strand) or reverse (and recognises the sense strand/ genomic DNA), I can't see how the target strand can still have two cytosines (or more) in a row. If methylated, it must be in a CpG dinucleotide and therefore would be either followed or preceded by a C in the primer. Furthermore, the preceding or following nucleotide, if a cytosine in the original sequence is therefore NOT in a CpG dinucleotide and would therefore be converted to uracil by the bisulfite treatment and hence, be correspondingly represented in the primer by an A.

I know which sequences we want to examine and am aware of principals of primer design and resources out there to help with this, but this question above has started to bug me and makes me realise I clearly don't understand a fairly basic aspect of this process! I am working with an experienced geneticist, although she has not done any bisulfite work and can't answer this query.

Any advice greatly appreciated.

Al

Question: "why primers listed in these papers (whether methylation specific or not) can ever include two (or more) G nucleotides in sequence?

Answer: Let's assume you are talking about primers for MSP.

For forward primer, it is easy to understand that one or more G in the primer is OK because almost all methylation occurs to the C in the CpG dinucleotides. But the primer should also contain at least one CpG C at the 3' end. For the M primer, the C remains a C; for U primer, the C is replaced by a T.

For reverse or downstream primer, two consecutive Gs do not make sense. Given the following sense DNA sequence:

sense DNA: 5' - TTACGCGCCTGGACCAGCT -3'
||||||||||||||||
bisulfite mod DNA 5' - TTACGCGTTTGGATTAGTT
||||||||||||||||
reverse M primer GCGCAAACCTAATCAA -5'


For reverse U primer, there should be no G at all.

Does that make sense?

Thanks for your reply.

I'm still confused I 'm afraid. Although what you've said is entirely consistent with publications I've read, where consecutive G nucleotides occur in the forward but not the reverse primers.

What does the forward primer bind to? I had assumed it was the antisense strand of DNA and that this would be subject to bisulfite modification in the same way as the sense one? Thus all the consecutive Cs (to which the primer Gs can bind to) would be converted to uracil. Is this not the case?

First it is assumed that DNA methylation is symmetric on both strands, so we will only consider the sense DNA strand here. During PCR, the reverse primer will first bind to the sense DNA and amplify it. Following that in the 2nd cycle, the forward primer will bind to the newly amplify DNA.

Excellent. That makes sense now. Although I thinks this definitely counts as falling at the first hurdle so sure there will be more stupid questions. Thanks for your help.