Different primer optimalization for nested vs direct MSP? - (Aug/13/2012 )
I am currently trying to optimalize primers for direct MSP. I started with a temperature gradient (58C - 64C) and 35 cycles. However, I did not see any bands, only primer dimers... The amplicon size is small (~80 bp), so I checked the PCR products again on a higher percentage gel, for the dimers might cover the actual bands.
Running a 2,5 % gel for 45 min yielded in very very slight bands and very very thick and dark primer dimers.
To be sure, that I will obtain amplification with these primers and to see if it is worth it to continue optimalizing those primers, I performed nested MSP. A gradient with nested MSP and (un)methylated controls worked well.
For I am going to use fresh-frozen tissue material, I would like to get my primers working also for direct MSP!
What can I try to get my primers working? Can I change the number of cycles with such a short amplicon size?
Looking forward to your ideas/experience/expertise!!
Do you do nested MSP using a universal primer set first and then MSP primers?
Some MSP primers have inherent defect and are beyond optimization, new primers have to be designed.
Because you are doing MSP, I won't trust any bands beyond 35 cycles.
Have you tried hotstart taq, such as Sigma JumpStart RedTaq?
Usually we design primers for a nested MSP, so a flank primer set and two 'inside' MSP sets (one methylation specific and one unmethylation specific). Those sets usually work well for cell line material and FFPE material. However, for fresh-frozen material we use direct MSP (MSP without prior amplification of flank region), for nested MSP is too sensitive and results show more false-positives. Therefore, we indeed use the same primers as for the 'inside' MSP to perform direct MSP. Usually we lower the temperature for a direct MSP to 2-4C lower. So if we perform a nested MSP with an annealing temperature of eg 66C, the same primers may be used for a direct MSP at the same, but more likely at lower temperatures, such as 64C or 62C.
What do you mean with inherent defects? And why would you not trust any bands beyond 35 cycles? I do not use any MSP primers with less than 35 cycles, but I know a few people who do.
We use Bioline Immolase Taq.
Thanks a lot for you help!
For nested MSP, I think it is more appropriate to use universal outer primer set to first amplify bisulfite modified DNA regardless of methylation status. Then the same PCR product can be used as template for nested PCR using either the M and U primers. Nested MSP using the same M or U set will likely introduce huge bias upon two rounds of amplification if they have different amplification efficiency.
By inherent defect, I mean bisulfite PCR primers are not and sometimes cannot be optimized by the criteria of normal PCR.
Regarding cycle number, similar to RT-PCR, you depend on the intensity of resulted bands to judge the degree of methylation, or expression as for RT-PCR, a high cycle number tends to give you false positive results.