Bisulfite sequencing of PCR products after AIMS technique - (Apr/20/2007 )

Hi all,

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?

Michiel

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...
Krümel

Michiel,

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.

Good luck.