Problems in bisulfite sequencing reproducibility - (Jul/12/2004 )
I am having huge problems with bisulfite PCR.
Southern analysis indicates that my DNA is unmethylated. My first bisulfite treatment also indicated that the DNA was unmethylated. Subsequent treatments have not been able to reproduce this result with all CpGs now being methylated.
I make fresh reagants each time.
Do you mean that your frist bisulfite sequencing results showed unmethylated DNA from treated cells and then you did another treatment as the first time, and the DNA remained methylated?
Are you sure your first sequencing results show clear and complete unmethylation and second shows methylation? Is the treatment condition the same?
If the answers to the above questions are yes, it is unlikely to get different results on bisulfite sequening which I think is more reproducible than MSP. Since it amplifies both methylated and unmethylated alleles, no matter how many cycles you use, the amplification of both alleles should be proportional and can be reliablely read out on the chromatogram.
You can do a TA cloning of bisulfit PCR product, and then pick ten clones randomly to sequence. It will offer more accurate data.
is your sequence being completely converted by bisulfite? As in are you getting Cs only at CpGs or somewhere else too? Make sure your bisulfite is not off, as well as other reagents, and obviously use the same protocol.
If you are talking about the same DNA sample that's the only thing I can think of. I agree with pcrman in that cloning and then sequencing clones might give you a better idea of what methylation frequency of your sites is-sites can be methylated or not within the same cell population.
Or did you extract a second lot of DNA and then bisulfite modified that? Methylation can occur spontaneously over time on cell culture.
I am doing Methylation sensitive PCRs, Modified template works sometime for PCR amplification but sometimes not? does anybody have a clue?
Thanks in advance
I have same problem with the guy, same time I can not repeat the result. I did very careful same condition but the result still can not stable. I used ZM Kit treated rat DNA.Thank you if I can received your advice.
Most people I have spoken to say that methylation specific PCR is all artifact and I tend to agree. I suggest doing sequencing of PCR products of bisulfite-modified DNA. This also gives you more CpG sites to analyze as opposed to just the ones in your primers. If you want a good protocol, see under my "the actual bisulfite treatment" thread.
One other thing I found that could be affecting your amplification is primer design.
Ideally you would want to design primers that selectively amplify fully converted bisulfite templates.
Parameters that I have used are based on Warnecke et al 2002 published in Methods.
I have found some online bisulfite primer design for bisulfite sequencing have not been optimal in amplifying fully converted DNA. That is to say I observe unconverted cytosine residues. You many have better luck redesiging your primers to another CpG site that will favour fully converted template