I'm doing a PCR for detection of Mutation on position 167 in beta tubulin.
It is a Nested PCR which a second reaction was performed using the primers Fs + R2 (141 bp) and F1 + Rr (212 bp). The Fs primer was designed to anneal only to TTC (wild type allele), while the Rr primer was designed to anneal only to TAC (polymorphic allele).
Also, they have mentioned that " Both primers had a mismatch at position 4 at the 3end to ensure high specificity. "
I got a band in mutation allele but i never saw a band in a WT. What is the problem? can someone help me?
Thanks
It is hard to say without seeing the sequence and the primers. The second change near the 3' end will make 2 changes in the unwanted allele amplification so annealing of the primer would require a very low temperature so you are less likely to get a product with the unwanted allele. You need a good quality WT dna that amplifies with any other primer set to show that it has no pcr inhibitors. Then run a temperature gradient of annealing temperatures for your primers as a start to troubleshooting. It is possible that yoyr WT target dna has a polymorphism in which case it may fail to amplify or that it contains pcr inhibitors. In this case you should select a low annealing temperature and pcr dilutions of 1:2, 1:4 ......1-16 because often if there are pcr inhibitors less dna means less inhibition and more amplification
Paul Rutland for one of my samples i got bands for both wild-type and Polymorphism. Can i say the sample is heterozygous?
- Badges
- Science method