I am amplifying a specific region of virus genome (650bp) with degenerative primers. my primer set, worked for 6 samples but failed for other other 28 samples. I used 55C the annealing temp. I believe PCR was positive for 6 samples, means PCR reagents had no issue, how can i troubleshoot with my primers?
Plz suggest some suggestions.
Dear Kishoree, It is quite common, I experienced the same issue for antibiotic resistant genes, phenotypic resistance was present but I was unable to detect the resistant genes for various antibiotics by PCR, what I figured out was that the sequence of primers I was using might not match with the target genes in bacteria (due to rapid mutations sequences change).
For your case it might be the same, possible solutions are either you have to increase of set of all possible primers complementary to the gene you are looking for or find a way to isolate the gene or look for the all sequences available in NCBI and request the Sigma company to design the primers of your interest, that can possibly help to identify the exact sequence you are looking for.
This is because of the rapid mutations in the regions where your primers anneal within specific 650 nts target region. To solve this, I think you should collect all the available nucleotide sequences for your interested region from NCBI or other databases and then you should do a multiple sequence alignment to find conserved sequences in the flanking region (both upstream and down stream) of your target sequence to design new set of primers which should work with all your samples unless there is any mutation present that is not identified or submitted to the database yet so far. Best of luck.
You should know the sequence of your target gene for designing new primers.
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