You could just amplify across the duplication and separate the alleles either on agarose gel and sequence the different size bands or clone the pcr product and sequence clones but about a 14bp difference in a pcr product of about 100 bp should be separatable on 2% agarose if run slowly at a low voltage and preferably in a cool room.

You might be able to see this on Sanger as a "messy' region starting at the known 2026bp location. Design primers that are at least 100 bp upstream and 100 bp downstream of the duplication.

Or you could use melt-curve based genotyping. Design primers that closely flank your known duplication site, include a full set of controls (known wt variant, known heterozygous variant, water, etc.). Include a fluorescent dye into your PCR and amplify like normal. Then, instead of an agarose gel, use a qPCR machine to run a melt-curve on your product. With an 18-bp difference, you could probably use a fast 0.5*C step melt and not high-resolution melting.

Good luck!

Hi Ashis Kumar

why not choosing one of the primer among the set across the duplicated/derived sequence?

I would do the same thing on the wild sequence and test the 2 distinct PCR with an internal control to test genotyping protocol as ARMS-PCR? it should be strictly specific.

fred