Posted 02 November 2004 - 11:43 AM
Posted 02 November 2004 - 01:09 PM
Posted 03 November 2004 - 06:55 AM
Posted 04 November 2004 - 06:29 AM
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