what is the effectors of MSP? - (Jun/10/2013 )
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,
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.
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.
Then, you can try optimizing PCR conditions to get consistent results, BTW, what is purpose of bisulfite treating PCR product - as a positive control?