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Problems with bisulfite modification


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#1 peaselab

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Posted 02 November 2004 - 07:40 AM

Hello,

I'm new to the forum and new to studying methylation patterns of genes.


I purchased the EZ DNA methylation Kit for bisulfite modification, but preliminary sequencing PCR products generated from modified DNA indicate that the modification process is not working at all. There do not appear to be any converted cytosines, even when they are not CpGs. The genomic DNA I'm trying to bisulfite modify was generated using phenol/chloroform extractions followed by EtOH preciptitation, so I thought the problem might be that the DNA is not clean enough. So, I tried modifying some plasmid DNA which should be really clean, again, no cytosines were converted.

Am I misundertanding how this process should be working? Should I dump this kit and try another?

If this is a matter of preferential amplification of unconverted DNA, what can I do to improve the efficiency of the modification process?

Thanks.

#2 pcrman

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Posted 02 November 2004 - 08:11 AM

Hi,

I would like to know how you designed your primers, can you post your primer sequence here?

Incomplete conversion is always a problem especially in GC rich region. Even a paper published on Nature mistakely claimed that as non-CpG methylation because they have used primers that was unable to tell converted and unconverted DNA apart.

It may help to digest your DNA near but not within the amplified region, or to ocasionally raise the incubation temp. to 94C degree during the o/n incubation.

#3 peaselab

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Posted 03 November 2004 - 08:17 AM

Thank you, pcrman.

I realized yesterday (after I posted my question, of course) that my primers are not designed to select for converted DNA. I'm in the process of designing new primers.

Along that note, I used the MethPrimer program to pick new primers within my region of interest. Is there another web-based program to design primers that you suggest? Do you suggest nesting primers as well?

#4 pcrman

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Posted 03 November 2004 - 09:16 AM

Yes, you can always use nested primers for sequencing PCR or MSP.




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