***In Silico PCR producing output for incorrect sequence*** - (Aug/28/2013 )
I need your assistance:
I am using UCSC's In Silico PCR to test primers that have been designed by a previous student. I should amplify a partial region on chromosome 7 (gene TAS2R38)
The designs are:
F: 5’- CCT TCG TTT TCT TGG TGA ATT TTT GGG ATG TAG TGA AGA GGC GG -3’
R: 5’- AGG TTG GCT TGG TTT GCA ATC ATC -3’
However, when I plug them in, the output suggests that it amplifies a region on chromosome 22. Can anyone explain why I'm getting these results?
Thank you kindly.
When I try them out, they amplify exactly what they should ... However, there is a mismatch between the forward primer and the template at the 3' end (the penultimate base), so I doubt whether these are ideal to amplify the wild type allele.
This is the sequence of the resulting amplicon (I removed the last two bases from the forward primer):
>uc003vwx.1__TAS2R38:185+404 220bp CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGC AGGTTGGCTTGGTTTGCAATCATC CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCaggcactg agcaacagtgattgtgtgctgctgtgtctcagcatcagccggcttttcct gcatggactgctgttcctgagtgctatccagcttacccacttccagaagt tgagtgaaccactgaaccacagctaccaagccatcatcatgctatgGATG ATTGCAAACCAAGCCAACC
Thank you dpo!