I was designing Tri-ARMS-PCR primers for an intronic variant of PUS10 human gene located at minus strand in chromosome 2.

When I run in-silico PCR for forward and allelic reverse primer i give specific results. but when i use another reverse primer of same gene with this forward primer it shows an error that no matched found.

I tried to find the sequences of primers in gene sequence on NCBI and it gave perfect specific match. by why in-insilico is not working for reverse 2 which is almost 600 bases apart from forward primer.