Advice on optimising bisulfite PCR sought - (Sep/28/2011 )
Looking for some general advice. I am currently working on methylation analysis by both MSP-qPCR and bisulfite PCR sequencing. The former appears to be working, but I am having problems with straightforward bisulfite PCR with almost all my primer sets, specifically seeing bands around 100bp (? primer-dimers, ?primer mispriming). These vary in intensity, but are usually stronger than the target band, which is there too. I was originally using published primers but tried redesigning them with methprimer without a great deal of additional success.
The aspect I really don't understand is that the published primers originally worked fine but then developed this problem after 5-10 experiments. So I replaced everything in case I'd contaminated something without success.
Nested PCR gives very clean bands - the same set of nested primers on converted DNA (without any prior PCR) give the usual smudgy bands at 100bp. This has allowed some sequencing and to generate standards for the qPCR. However, I am still puzzled as to why the 1st PCR has this systematic error.
I have tried a variety of strategies - altering substrate DNA amount, primer, magnesium concentrations, annealing temps, extension temp, tried different machines. I am using promega hotstart Taq. The target sequences are between 300 and 500 bp (all the same area but vary with primers). My program is as follows: (these are from published literature)
95C x 10mins
95C x 1min
55C x 30s (this varies)
72C x 1min
72C x 10mins
I realise that there are a number of possible strategies to try now but wondered whether anyone had any useful starting points. Would trying a different Taq be worthwhile? (I have tried the qiagen one too without success). Would addition of glycerol or DMSO add anything? Would a touchdown approach or shortening cycle times be worthwhile?
Any advice gratefully appreciated.
we've found that the taq makes a big difference. our current favorite is the Epimark hot start taq from NEB. They also recommend an extension temp of 68C.
Also bisulfite DNA is not very stable so avoid many freeze thaw cycles, we keep ours in the fridge for up to one month or freeze thaw 4-5 times only. The conversion kits also appear to have different efficiencies. we find bisulfite DNA from the zymo 96-well plate terrible.
Worth a try - thanks.