Bisulfite sequencing problems - T>C conversion (Mar/14/2005 )
i think you may be dealing with a simple artefact. To rule this out, repeat the genomic DNA isolation (If you can) and ensure you proteinase K treat the DNA. Phenol Chlorofrom extract twice and the chlorform extract to remove traces of phenol and then isolate the gDNA by ethanol precipitation. For bisulfite treatment you should ensure that the gDNA is properly denatured, for my old protocol you add to the DNA a final concn of 0.3M NaOH and you incubate for 15 minutes at 37C, I add a step of 100C for five minutes before immediately adding the bisulfite solution, there is another denaturation step and I repeat the same process as stated above. If you are able to repeat all this again and the conversion is not seen, then it is artifact...
One other thing I have just thought of, is your primers are they strand specific, because if you picked primers as if it were a conventional pcr, you would run into real problems.
I agree with Nick. He emphasized an important issue: using very pure DNA for modification otherwise DNA may not be completed converted.
I was reading your email responses regarding bisulfite sequencing. I use chemicon kit for bisulfite treatment of my DNA. I have trouble amplifying larger PCR products or seq spaning more CpGs. I read your message and you seemed to successful in amplifying a 600 bp DNA with 60 cpGs. It would be of great help if you could give me more details on how did you get it to work.
I have sequenced, after bisulfite (CpGenome, Chemicon), first PCR: 45 cycles, second nested PCR 30-35 cycles (SigmaREDTaq) and subsequent cloning, a 635 bp product, containing around 60 CpGs. Sources are different genomic DNA samples.
Although my results are negative for hypermethylation , I'm stuck with the following. In some of my samples (not all) I find a T>C conversion after sequencing. There is a clear preference for the location of this conversion (a total of 4 locations, although the locations are shared in different samples, not all combinations are the same).
Is this an artifact? (Due to 2x PCR? it occurs also in a few samples which I cloned without nested PCR) (Due to bisulfite? it doesn't occur in all samples treated at the same time).
A suggested mutation in the genomic DNA does not make sense to me, since normally this cytosine would have been converted after bisulfite (I'm now anyway trying to sequence directly).
I would be grateful to all suggestions.
to amplify greater than 600bp of sequence you should design a two-step PCR using nested primers. The first primer set can amplify up to 1kb of sequence and the internal nested primers should be picked to amplify greater than 600bp of sequence.
I typically select primers that are 30mers in length to ensure a high Tm.
all the best and if you need more help,you'll know how to find us!