all the cytosines are converted to thymine? - (Sep/15/2005 )
i treated the DNA using the protocols just like the onlines(my objective is a rice gene's promoter island), but after the sequencing, i found all the cytosines are converted to thymines, i con't believe that, anyone can tell me, why?
i found in some other protocols, they all treated the DNA in 50°C, but the protocols online treated in 55°C, can anyone tell me the difference?
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I take it you are looking at a CpG island within the gene promoter? If so, CpG islands are usually unmethylated and all C's will become T's, it's an expected result. Not much around is on plants (I haven't really looked) but I would bet that CpG islands in plants will behave the same as mammals (please correct me if I am wrong) in that they are mainly unmethylated.
I am not too sure if there is any non-CpG methylation due to a Chromomethylase like that found in Arabidopsis, is there a orthologue in rice???
As for the differences in temperature, I have stuck with 55C with no problems, I have also tried 50C, same deal, I recently came across a paper describing how bisulfite conversion can be achieved at 95C within an hour or so, I think the high temperature ensures that the DNA is in a single-stranded state for bisulfite reaction.
See full conversion is a good thing!!!!!
I agree with Nick. I would first consider it as zero methylation. Over conversion (5mC -> U) is although possible, the rate at 50-55C is very low.
How do you know all cytosines are converted to T, by direct sequencing or cloning? If it is the formmer, have you noticed any small cytosine peaks?
Hi, thanks a lot for your reply!
The gene be working on is about photosynthesis in rice. In different parts, it has different expressions. I thought that the methyl-C's in the promoter of the gene may have some differences, but in my study, there have no differences in the first CpG island (about 500bp long, most close to the ORF) even in the roots, all the cytosines are converted to thymines.
Usually, there have more than one islands in the gene’s promoter region, can you tell me which island is more important for the gene’s expression (both in plants and mammals)??? Certainly, eliminate the motif effect.
By the way, I sequenced it by cloning.