Gregor Bucher wrote:
> Does anybody has experience with allele-specific PCR. I´d like to have
> some hints how the primers and conditions should be chosen before I start
> to find out these things again. To my knowledge only A-G C-G and A-A
> mismaches will give results.
>> What else can I do to make the reaction work?
>> Thanks a lot!
I'd recommend you try and find a very useful book called "Finding
mutations:the basics" by J.R. Hawkins, Bios Scientific publishers, ISBN
0 19 9636117. This has a short chapter on the theory and practice of
ARMS/allele-specific PCR that includes recommendations for adding
appropriate destabilising bases next to the actual mismatch to extend
the technique to all possible mismatches.
In my experience, varying the concentration of the allele-specific
primers from a 'typical' PCR concentration usually makes the assay work
correctly, though this can take a lot of work to get right as it is
often quite a narrow range of concentration. Obviously, good controls
for the different genotypes are essential to test this.
Hope that helps,
Dr Andrew Walley,
Asthma Genetics Group,
Wellcome TRust Centre for Human Genetics,