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Strange results from bisulfite sequencing - (Jul/31/2009 )

I'm new to methylation techniques so please excuse any apparent ignorance on my part!

I used the EpiTect bistulfite kit from Qiagen to modify my DNA, and I used PCR primers designed with BiSearch to amplify a section of SNRPN in the human genome. The ultimate goal is to be able to detect Prader-Willi and Angelman syndrome with this PCR/cycle sequencing assay. In theory, a normal person would have one methylated copy and one unmethylated copy of the genes in this region, thus producing heterozygosities in certain base pairs. A mutant individual would be homozygous (I guess technically it would be hemizygous) for those base pairs (the mutation I'm trying to detect is a large deletion of one of the alleles).

I tested the protocol with my DNA as a control and I got some rather strange results. There are several A/G heterozygosities (I'm sequencing using the reverse primer, so A/G makes sense) but I'm also seeing several C/G and T/G heterozygosities as well. I ran the PCR product on a gel and it looked clean (one band and no smear of non-specific amplification) so I don't think I'm sequencing another similar gene in addition to the one I want. I read somewhere that some sequence analysis programs will notice the lack of a certain dye and therefore artificially increase the response when it does see it. Could these unexpected heterozygosities be tiny bumps of noise that the program is artificially increasing and calling heterozygositeis (I'm using SequenceAnalysis 5.2 by AppliedBiosystems).

I can scan and attach a snippet of the sequence if you want to look at the actual electropherogram.

Thanks for any help!


Please paste your chromatographs here so we can get an idea what is wrong.


Here's a 2 page PDF.

The first page is the reverse sequence that I mentioned in the origional post.

After I posted the first time, I sequenced with the forward primer and got very few C/T heterosygosities, and the two that I got (on the bottom line) look somewhat questionable.

Any ideas what might be going on?
Attached File