methylation Primers - Methyalted PCR (Dec/17/2005 )
this is my question: when your making methylated(forward and reverse) and unmethylated(reverse and forward) primers, there is no way to work back to your wildtype primer? ive figured this is due to the changes of the selected nucleotides, so how would you then work back to the origional strand if you were given the methylated and unmethylated primers? Ive tried to do this to ensure the primers ive located through the literature ive read is correct, but if i as a researcher cant trace back to the origional strand how can i be sure the primers given are correct? Im sure we all know that we need to check up on the sequences off primers as a simple misprint in journals is very possible. so could someone explain how i could check these methylated and unmethylated primers are occuring from the target sequence i require?
thanks for the help and advice in advance. And seasons greetings to you all
I would do a mock bisulfite conversion, if you know what gene/sequence you MSP primers are directed to, download the sequence of interest and in a word processor, find and replace the nucleotides assuming 100% conversion. That is, firstly maintaining all the CpG dinucleotides, change these to say NG, then find and replace all C's to T's, and Find and replace all NG to CG....now you have converted your DNA sequence, you should be able to then search for your methyled primer sets and you unmethylated sets should be in the vicinity of your methylated set, if you can not find it, reduce the stringency.....decrease the length of the primer until you find you sequence, no match equals not the primers!