Allele-specific DNA methylation - What to do if the discriminating SNP is a T/C ? (Jan/29/2007 )

Hello everybody,

I think I've got a tricky one today:
We want to analyse the DNA methylation of a certain gene with respect to the genotype. I think, we need to do the analysis allele-specific. The methods described to do this are all discriminating between the alleles by using SNPs in the binding region of the PCR or Sequencing primers.
My problem is, that the SNP I am interested in consist of C --> T. So if I was using bisulfite, all alleles would become identical
Is there a way of separating the alleles without destroying the methylation information (i.e.PCR)?

Any suggestions are appreciated!

Krümel

Hi Krumel,

I would say you can design primers that specifically amplify the opposite strand because C --> T in the + strand would read G --> A in the - strand and will not be affected by bisulfite conversion.

Good luck!

Nick

Thanks Nick, that sounds reasonable. I knew I was missing somehing...

Krümel

Another question,

in which step would you introduce allele specifity? I was thinking about a common first round PCR and afterwards allele specific primers, but I have also seen a paper using specific primers in the sequencing reaction. Any opinion?

Thanks,

Krümel

I have seen papers where thye just performed BSP, cloning and sequencing and highlighted the SNP within the lollipop figures.

This would be easier as there is no need to optimise allele specific primers and you get your methylation data surrounding it.
N

I guess you're right.

I will try that.

Thanks, Nick!