allele specific pcr - determining genotypes (Dec/12/2009 )
I am currently doing allele specific pcr (AS-P) for identifying SNP of an individual and hence the genotype . I have designed primers wherein there is last base change at the 3'end of the forward mutant primer i.e the polymorphic site and an additional mismatch created at the pre-penultimate base so as to increase the specificity amongst the primers and the template. The problem is i am not getting any of the homozygous mutant. I can differentiate individuals with homozygous wild and heterozygous. while designing a pcr, i am setting up two reactions; one with forward wild- reverse primer addition and other with forward mutant and the common reverse primer. when an individual is homozygous Wild, then there will be amplification in wild tube and amplification in only mutant tube will indicate homozygous mutant. So how to get rid of an amplification of the wild type primer even though i know the sample is homozygous for the mutation.
for the information Tm for forward wild = 60.3 degree celsius
forward mutant = 56.5 degree celsius
common reverse= around 70 degree celsius
primers are annealing at 55 degree celsius
waiting for an early reply.......
I seriously don't think a single mismatch can differentiate SNP in PCR. You need to use specific probes such as Taqman, or go by High Resolution Melts.
A single 3' primer mismatch will generally prevent PCR amplification.