We are trying to identify single nucleotide polymorphisms (SNPs) using allelic discrimination software on the Roto-Gene 3000. Each reaction includes a wild type probe, mutant probe, and DNA template with a known genotype at the SNP location. We have been fairly successful in differentiating wild type and mutant DNA templates. However, in the reactions containing heterozygous DNA template the wild type probe signal is much higher than the mutant probe signal, leading to the heterozygous control DNA to be called as wild type. Either the binding of the wild type probe needs to decrease or the binding of the mutant probe needs to increase.
Does anyone have any suggestions on how to improve our results? We have tried varying the concentrations of the probes without much success.
Thank you!
Hi Molly,
Without observing the amplification profiles, what you have explained appears typical of a genotyping assay. We run several genotyping assays and rarely do we observe identical amplification from both dyes in the heterozygous profile, which can be influenced by the different dyes on both probes and reaction efficiency. I am not familiar with the rotor gene software, but it is important that the clustering from the allelic discrimination plot is not ambiguous. If so, then the assay is probably working fine. We then use the amplification plot to ensure that the profile is consistent for all samples in that genotype cluster.
We are able to manually re-call the genotypes within the software, if the software has miscalled, which may be an option for the rotor gene.
I have attached some profiles for one of our genotyping assays that may show similarities. This assay works very well despite the amplification differences in the heterozygote profile. When we call our heterozygotes we ensure that the blue curve (FAM) is consistently stronger than the green curve (VIC). If this deviates we do not accept the result and will iether sequence or use a RFLP assay to discriminate the genotype.
Brendon
Hi Moly
If you want to do it in a fast and cheap way look at my two papers which are uploaded by me.
Hi Moly
If you want to do it in a fast and cheap way look at my two papers which are uploaded by me.
The signal from amplification also depends on the fluorescent dye used for each allele. FAM usually give higher fluorescence than Hex/ViC/TAM and therefore, the allele using FAM will show better curves than the other dyes. Is your Wt allele labeled with FAM?
@Brendon O'Rourke Thank you for you advice! The pictures you have uploaded are exactly what is happening. Unfortunately the software calls the genotypes automatically and the samples cannot be relabeled in the case of a miscall. You answer gives me hope that our results may be okay to use.
@Omaththage P Perera Thank you for the information! We are using FAM to identify our wild type allele and HEX for our mutant allele. Perhaps I will look into ordering a probe with a different dye for the wild type.
Hi Molly,
Primarily our taqman assays are for inherited disease. As a rule, we use the FAM dye for the minor allele (disease allele) because it is a stronger dye.
Good luck with your assays.
Brendon
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