Problems with MSP - Unable to pick up bands for methylation (May/23/2005 )
I am trying to look at the methylation status of three genes of the murine MHC class I - Q9, Kb and Db using MSP.
The primers for Q9 work well and I am able to pick up both methylated and unmethylated bands in tumour and spleen samples respectively. The sequencing data show efficient bisulphite conversion too.
But for Kb, Iam just not able to pick up any band at all.
I have tried primers with all possible permuations of methylated and unmethyalted CGs to no avail.
Could anyone give me any ideas on what iam doing wrong/ what could be going wrong?
The Tm s for my Kb primers are in the range of 46-52 C and the polymerase I am using is Qiagen's HotStarTaq which extends at 72 C. I do get good bands for Q9 ..whose primers have Tm s in the range of 52-57 C with the same polymerase and under the same conditions.
Do u think it would be a better option to go ahead and design primers with no CGs at all?
Any suggestions would be really appreciated.
You have one pair of primers that works, which is good sign. At least your DNA is good. The problem may lie in the primers. I would suggest that:
1) You didn't mention how many cycles you run your PCR. You can extend to 38-40 cycles.
2) If that doesn't work, redesign another pair of MSP primers
3) You can also design nested primers with the outer primers containing no CpG sites (Universal), and the inner primers being MSP and USP pair.
Since you are doing MSP, how can use avoid CGs in the primers?