I am trying to genotype SNPs by using two multiplexed probes: one with the mutant DNA sequence (HEX - 3BHQ_1) that fluoresces on the green channel and one with the wild type DNA sequence (6-FAM - 3BHQ_1) that fluoresces on the yellow channel. I did this successfully with one set of probes and allelic discrimination software. However when I attempted the same protocol with DNA with a known genotype and another set of probes for a different SNP I saw fluorescence in the incorrect channel. By this I mean wild type DNA showed fluorescence in both the yellow and green channels. Previously with the first probe set I saw wild type DNA only fluorescing on the yellow channel. Does this suggest a lack of probe specificity? Do you have suggestions on how I can improve the specific binding of the probes?
Thank you!
Yes, this is a lack of probe specificity. Unfortunately we see this sometimes in pre-designed SNP assays sold by Applied Biosystems. Of course Applied is blaming the customer but I know several groups using different cyclers and kits who obtain the same unspecific results.
We have tried several changes to improve the specificity but there exists no common solution. End point discrimination is not feasible, the only possibility is a look at the ascending slope to discriminate WT and Het. In this case you need on each plate sequenced controls for the classification.
Hello Molly
We often had the same problems with unspecific binding. To my knowledge SNP genotyping by ABI is still done by real time PCR, so try to play a little with the annealing T and/or concentrations no matter what the protocol says. In ABI cyclers you can also use different sets of annealing T within the same run by dividing the cycler block in different PCR sets. For this I suggest to use different DNA probes with different SNP genotypes, wildtype and variants (if you don´t have or know it just sequence the SNP area before) and try and see what PCR conditions work the best.
I hope this might help a little.
You may redesign your TaqMan probe to improve specificity. First try on standard control.
We usually have success by increasing the annealing temperature to 62 degrees. We also use the SNP genotyping master mix which gives better results than the universal master mix.
you can try primer & probe conc. gradient & even if it doesnt resolve try annealing temperature gradient, also try to reduce annealing time.
Hello Molly
I just wonder whether your problem has been solved? I have met a problem similar to yours. I have no idea where the problem is?Maybe the reaction mix?Maybe the Taqman-MGB probe ifself?
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