Having difficulties with your experiment?

We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios with your drug metabolizing enzyme (DME) genotyping experiments.

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General

Depending on the assay, you may want to try either reducing or increasing the number of cycles. Our newer instrument software can even allow you to view the traces if you collect the real-time data on the instrument.

Check the Minor Allele Frequency (MAF) of the SNP. You may need a larger sample size in order to see the allele. You can use the Hardy-Weinberg equation to determine if the minor allele is detectable in your sample size or not. Follow the example here (p. 4-2).

The software does not allow assay IDs or sample IDs to be modified. If a typo occurred or an edit is required, this change must be made in the original experiment file. The software does, however, allow you to create your own assay name and add additional information related to an assay or sample through Setup -->Assays or Setup -->Samples. 

TaqMan® SNP Genotyping Assays are an ideal technology for interrogation of most DME and clinical research target polymorphisms, offering highly specific target amplification and allele discrimination. However, there are some polymorphisms that are not well‐suited for TaqMan® assay development. These include targets that: 

  • Reside in highly polymorphic genomic regions (polymorphisms interfere with amplification in some samples) 
  • Share high sequence identity with another genomic region (base differences are not available for specific assay development)
  • Are microsatellite polymorphisms, or 
  • Are base deletions within a homopolymer sequence. A list of commonly requested DME and clinical research targets that are not good candidates for TaqMan® Assay design and suggestions for alternative technologies to use are included in the PGx Common Markers file

If you are not getting calls in the instrument software, you can try the free TaqMan® Genotyper Software. This program has an improved algorithm which allows it to make calls that are often missed by the SDS software. See the example below:

The same data was analyzed with either the TaqMan® Genotyper Software or the standard Auto calling with the instrument (7900) software. While the data has a problem with trailing clusters, there are clearly 3 clusters present. The TaqMan® Genotyper Software is able to call the majority of the samples, while the Auto calling cannot.

In addition, make sure you are running a sufficient number of samples (more than 3), and include at least one NTC well which is marked in the software.

 

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