Bisulfite sequencing of PCR products after AIMS technique - (Apr/20/2007 )
I am new to this forum and to the research field.
I used the AIMS technique (Frigola et al. 2002) to amplify methylated sections of DNA. This is based on restriction digests, with linkers ligated to methylated restriction sites, after which a PCR with primers designed for the linkers gives amplification of methylated sections.
To confirm the methylation, I should do bisulphite sequencing on the amplified bands.
After a quick literature search on bisulphite sequencing, I found everyone start from genomic DNA to do the bisulphite treatment, after which a PCR with specific primers for the sequence of interest is done.
I wonder if I could do the bisulphite treatment on my original PCR product from AIMS, so that I could do the amplification of the bisulphite treated sample with the same primers (adapted for the C=>U conversion in the linkers) as in the AIMS procedure?
If you do bisulfite treatment on a PCR product, you're likely to get 0% methylation as a result, as PCR erases all methylation information...
Thanks a lot,
this question proves I'm a newbie
the thing to do after AIMS is to extract and clone your amplicons of interest for sequencing. This will tell you where in the genome it is by blast/'blat.
To confirm the methylation status, you would then go back to your genomic DNA, bisulfite treat and then design primers to those regions you have identified.