MSP gives smear only - (Oct/05/2008 )
I have been designing primers using Methprimer for MSP. The primers that I which are methylation specific work initially giving me my product but a bit faint or with other minor bands and then it completely disappears giving me just a smear.. Ive tried changing the chemicals, water, trying diff temp, Mg2 concs etc but all I get is a smear.... I just dont understand what is happening and itsdriving me nuts.... Ive tried repeating the initial conditions and yet I get just a smear and this happens only with the methylation specific primers and not the Unmeth primers.. Ive tried designing a new setbut I got the same result... COuld anyone help me out with this???
I have been reading the other posts in this forum and have found that heminested or nested PCR is adviced for MSP. I have been using the same primers for the second PCr.. COuld this be the reason for my problems?
I read the post about how to design heminested PCRs for BSP.. Can the same method be used for MSP?
I have a suspicion that maybe the primers are not good.... is there any other site besides Meth primer which I can use to design MSP primers or are there any companies which provide real time assays for MSP?
Please could someone give me the link to any post which gives the guidelines to design MSP primers. MAybe I have missed out on it...or if there is any specific paper which explains how to design primers as i have not much success in finding any such paper...could someone give me the papers details...
I would really really appreciate the help cos at the present moment Im at my wits end and have no clue how to proceed further.
thanks in advance
Have you tried hot-starting your PCR. You can do it manually and enzymatically. For manual hot-start, preheat your PCR block to 94C, then transfer your PCR tubes from ice to 94C. For enzymatical hotstart, use hotstart enzyme such as the RedTaq JumpStart from Sigma which makes very big difference compared to regular one.
For nested MSP, you need to have a pair of universal primers that only amplify modified DNA (including methylated and unmethylated) and are outside of your MSP and USP amplicons. To ensure that universal primers amplify methylated and unmethylated DNA without bias, they should not have CpG site in their sequence (although that cannot absolutely avoid bias).
Hope that helps.
For nested MSP, you need to have a pair of universial primers that only amplify modified DNA (including methylated and unmethylated) and are outside of your MSP and USP amplicons. To ensure that universial primers amplify methylated and unmethylated DNA without bias, they should not have CpG site in their sequence (although that cannot absolutely avoid bias).
Hope that helps.
THanks for the quick reply. I have been doing a manual hot start PCR so far. I ll probably try to get some of the RedTaq Jumpstart from Sigma. Maybe it will makje a difference.
Concerning Universal Primers, I was wondering if I should use ABI's Methylprimer Express to create them or maybe modify the DNA and use primer 3 to get me the primers.. What is your opinion?
Regarding outside universal primer design, methprimer can do it too. just check Design BSP primer. If you use Primer3, you have to tell it not to design primer on any CpG site. If CpG sites are unavoidable, degenerative primers then can be used to take care of potentially methylated and unmethylated CpGs.
Thanks a lot!! will do that!