You could try to do a long range PCR with a larger product spanning the SNP region, and then nest it with the degenerated primers. I am not sure if that would help but it may!

Hi Tania,

I would say that adding some informations would help community to advise you.

what is your purpose with this A/G SNP? in which species you're working on?

fred

Hi Fred,

Sorry, I thought I had given the needed details without overflowing the community with information. It is a thin balance… And gets even more difficult when you are in the middle of it! Please ask whatever you find helpful.

I am interested in genotyping this specific SNP as we know that it is, together with others, responsible for a specific phenotypic trait. I have good quality DNA of the insects, so that should not be a problem. I was just trying to find an efficient low cost way to detect the SNP, without having to go to sequencing, RFLPs or other more costly methods.

Thanks!

Hi Ana 

Thanks for the suggestion. I did not try that yet. I might though... However, I just fear on the specificity of the egenerated primers. I will let you know wether it gave any positive result. Maybe it works, but then the genotyping gets less cost eficient. :-)

Thanks!

Hi Tania

now I see the limits of your request. as I imagine that you have some degenerated genomes, and guess your difficulties to design primers, next to or away form your target, I would design an ARMS protocol which is efficient, cheap and rapid (one reverse primer, common for the 2 separated PCR with forward primers specific for both alleles).

hope this help

fred

HiFred,

Thanks a lot for your suggestion. I am not familiar with ARMS protocol, but from what I briefly saw it sounds extremely promissing! I will dig into the topic and will definitely try. I will let you know how it proceeds.

Once again, thanks for the help.

Tânia

Hi Tania

you're welcome...

fred