Success with Direct Sequening of Bisulfite Pcr Products - (Mar/24/2006 )

Just to let you know. ESME works relatively well. The problem with the free version is that you cannot change the reference sequence (which is human chromosome 6). If you want to look at other sequences, you have to buy a license which costs 2000€.

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Hi,nick
Could you introduce a literature about peak height scoring method ?
Can I use 4peaks with PC?If not,any alternative do you suggest?
Thanks a lot.

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sent you a paper fxs via PM.

As for PC chromatogram readers, you can try the staden package, ABI Sequence Scanner to name a few, also FinchTV does it


Nick

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Hi, nick
You are very helpful,thanks a lot. I've read the paper,and the method mentioned in the literature seems complicated. It seems hard to analyze my data.
As for quantification analysis of methylation, is COBRA a good choice?Can i use 2 or more restriction enzymes in a reaction?

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I don't think quantification by COBRA be a good choice, because you are relying on the efficiency of the restriction enzyme, something like methylight would be better and i would say it would be best to stick to one enzyme because your endpoint would not be able to tell which site was affected.

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Thanks a lot.I found that Cobra is a simple method analyzing methylation when methylation is obvious.It gives rough methylation status before time-consuming cloning.
Now I will perform Cobra before clone sequencing.

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I have a question about direct sequencing of BSP PCR products. It sometimes happened to me that, when I sequence my PCR product with both Forward and Reverse primers, both sequences are OK but methylation level is different; Most of the time, I observed methylation on one sequence (forward primer) but no methylation at all for the second sequence (reverse primer). Could it mean that my fragment is hemimethylated ?
thanks in advance.

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Ok, this may get confusing, but bear with me!

You begin with two strands of DNA, strand A and strand B. After conversion, your DNA strands are no longer complimentary. Therefore, your PCR is only targetting one DNA strand. Lets call this 'strand A'. Your PCR will target strand A and make complimentary copies of this to yield a double stranded DNA product, comprising of strand A and a newly synthesised strand, which we shall call strand C.

Therefore, to quantitate the methylation on strand A, the forward sequence will show C/T peaks at the C position of CG dinucleotides. In the opposite direction, the sequence will show G/A peaks in the G position of CG dinucleotides. This is because you are only looking at the methylation in the C position of the original A strand.

I think this is where you are getting confused - the C position in the reverse strand is always going to show C as it corresponds to the original G. It is the G position in the reverse which needs to be measured, as this is the position which corresonds to the C in the orignal A strand.

To show hemi-methylation, you will need to design another set of primers that will amplify the B strand.

I hope I could convey this clearly for you, it is difficult to explain!

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Well, I have a question here: seeing that the PCR and sequencing in BSP are both strand specific, Should we select the strand (+) to do so? Does the strand (-) gives the same informaiton about the methylation status of the gene promoter?

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Good question. Technically they should be the same, as DNMT1 methylates a newly synthesised DNA strand to make the methylation match that of its older partner. However it may be different if you are particularly looking at hemi-methylation. I have always targetted the sense strand of DNA for PCR, but for no real reason other than its easier for me to interpret the DNA sequence that way.

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