Duplex MSP? - (Aug/28/2007 )
Just wondering if any people have heard of this "Duplex MSP". I came across an article using this technique and was quite surprised that it actually works as the M and the U primers are placed together in one tube for the PCR.
The links for the articles are:
I read through the 1st paper and didn't really understand the MSP data presented. Can the experts here please do enlighten me about this duplex MSP?
Thanks and May the Methylation Force be with you...
If I got it right, they have been using primer pairs which produce different amplicon sizes. So you can either see one M or one U band. It may be more specific than regular MSP, where you use similar primers in different wells.On the other hand you are looking at slightly different fragments of the promoter, which could show different methylation.
Personally, I wouldn't use it...
MSP can be fiddly as it is, like with all multiplex PCRs all primers must be optimised in singleplex before multiplexing, PCR efficiencies for two primer sets in multiplex could be a whole new kettle of fish and give rise to erroneous results.
Krumel is right it seems the products are distinguished by different amplicon size and this could only occur if the primers are binding to different parts of the promoter of interest, so strictly speaking you can't really compare the two amplicons unless you assume the CpG's the primers all bind to are representative of the methylation status of the whole region.
Having said this, I can see how it could be a good screening assay if you have lots of samples and just want to quickly test the methylation status of one promoter quickly. Then you would invest a lot of time developing the assay, otherwise it's complicated!