I have done a lot of PCR to identify a mutation in beta-tubulin.
I have two sets of primers, one is for mutant and one is for wild type. I did nearly 100 PCR but I have a problem getting a good band in the mutant band. I got the strong band in the wild-type but in the mutant, my band is always faint.
Does anyone know why is this happening?
Something is amplifying better than your amplimer. Does tubulin have pseudogenes or is it part of a gene family with similar sequences that are amplifying. Did you use primer blast to check where the primers bind in the genome?
The expected band does look very weak...is it possible that the mutation is in linkage disequilibrium with a second change/polymorphism that leads to very weak binding of your primer.
If you post the primer sequences there is more chance that someone will see the answer
Paul Rutland
Thank you for your response.
The primer is related to codon 167 of the beta-tubulin which the mutation in this position is related to anthelmintic resistance. in some recent result there isn't any band near 500bp but i have still smear and weak bands.
I have posted the sequence of my primers for you and others.
F1: AAG AGG CGG AAG GAT GCG
Rr: GGT GAG GGG ACA ACA CAG T
Paul Rutland
I'm doing something similar to this paper
Article Molecular analysis of the F167Y SNP in the β-tubulin gene by...
Paul Rutland Is it possible that because of low-frequency of mutation i have gotten weak bands? as i got a band in both mutant and wild-type my sample is considered as heterozygous, so i'm thinking about this possibility.
If you have the mutation then a strong band is expected. Ideally you should have a dna control sample which does have the mutation as a control sample in your pcr assay. This would tell you that the mutation is amplifying so that you can have more confidence that any small amplified band is the right thing and also that no band is a real result rather than a failed pcr
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