allele-specific PCR - detection of SNPs by allele-specific amplification (Nov/02/2004 )
Hi! Can somebody explain me why by inserting a single mismatch - near the 3´-end- into the allele-specific PCR-primer the specificity of th e PCR can be improved?
I don't understand where a mismatch is inserted and why it is needed. For allele specific PCR, you just design two sets of primers with each for one allele and the nucleotide at most 3' end of the primers match one of each allele.
Practically I is acepted to insert a mismatch e.g. 1-5 base befor the allelspecific 3'end-base. It is observed that the amplifcation is more specific than without the artificial mismatch!? I is contrariety!?
the reason that introducing a mismatch in the 3' end increases specificity is the following:
The 3' end of the primer is essential in the extension of the primer, but if Tm of this part of the primer is high, even the mismatch from the allel you are trying to discriminate won't lower tm sufficiently to prevent the "wrong" allele primer to be extended. However, is you deliberately introduce an additional mismatch, the primer is more sensitive to yet another mismatch (from the variable allele) and hence will discriminate better.
I hope this explains,
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes
Sounds quiet logical!