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Nested allele-specific PCR - (Apr/10/2006 )

I am currently working on the development of an assay to detect a single bp mutation in genomic DNA. I've tried a number of strategies to make the assay specific and sensitive and my results boil down to the following:

Nested PCR greatly improves product yield.
After the first round of PCR, I use each amplicon for two reactions: WT and mutation-specific.
Both use the same reverse primer, but the mutant primer has a 3' match only for mutant as well as a mutation 3-bases in from the 3' end to further destabilize the primer.

The problem I'm running into is that I get too much non-specific amplification, even at 2mM MgCL and a (relatively) high annealing temp (62) for this primer set. I've used a number of Taq products, but the best (Roche Faststart) only slightly improves the non-specific amplification.

Any suggestions?



So one primer set contains the mutation while the other amplifies wildtype only? I would think that a single bp at the 3' end would prevent amplification in either case... maybe if you leave out the other destabilizing mutation that would help? I was doing point mutation and in my hands a single bp mutation, even 2 bp away from the 3' end prevented amplification while having 3bp between the mutation and the 3'end (also made the 3' end a G) did amplify...