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what is the effectors of MSP? - (Jun/10/2013 )

Hi,

I am a new person in DNA methylation. I have bought a bisulfite modification kit for test of DNA methylation. First I did a control test, that I treated a 400bp PCR fragment with several CG site, then i did the PCR using U and M primers. The results of MSP in two time MSP were different. I found PCR products in the U and M primers amplification at second time. what is the reason, it may be incomletely converion? anyone can give me a help? thanks,
Shanjun

-Sj Chen-

Quite interesting--you bisulfite modified PCR product. Did you purify the PCR product before the treatment. The inconsistency in repeats may be resulted from non-specific amplification of the primers. MSP is prone to mispairing especially if the CpG C is not at the very 3' end of the primer. In MSP, annealing temperature and cycling number are important and need to be optimized.

-pcrman-

Thanks. yes, I purified the PCR products and the M and U primers were designed by methprimer (plus CpG island prediction) of The Li lab.

-Sj Chen-

Then, you can try optimizing PCR conditions to get consistent results, BTW, what is purpose of bisulfite treating PCR product - as a positive control?

-pcrman-