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- Title
Validation of an Allelic Discrimination Assay for Drug Metabolism.
- Authors
Dablouk, Hatoon; Hendrix, Ericka; Nwokey, Chanel
- Abstract
The goal of this project is to validate a series of genotyping assays in order to investigate drug metabolism genes that may also be associated with trigger points and myofascial pain. We hypothesized that the allelic discrimination assay would be more advantageous for genotyping the candidate genes as an alternative to sequencing, which is currently the gold standard. Genotyping studies were performed using a sample size of 19 subjects. Subject DNA were previously isolated and de-identified prior to assignment. Genotyping was performed using TaqMan allelic discrimination assays (Applied Biosystems) on the StepOne real-time PCR instrument. To confirm the genotype determined, a percentage of the samples were analyzed by Sanger sequencing. The GenoTyper app was used for comparison to StepOne software for genotyping. We were able to obtain 100 % accuracy when comparing the allelic discrimination assay to the sequencing method. 100% reliability was obtained within assay for both SNPs with 100% reliability for the CYP2C9 SNP and 88% reliability for the CYP1A2 SNP between assays. We established our LOD at 10ng/μL of DNA. We could not confidently validate the assay due to the software’s inability to automatically call the genotype without manual assistance from the use of the multi-component plot. Hence, we were led to seek an alternative software to predict the genotype without manual interference. In conclusion, we have determined that the allelic discrimination assay validation had not been fully successful due to the software’s inability to accurately call genotypes. For future studies, we recommend the use of the GenoTyper (GT) app for accurately identifying the genotype due to its advanced algorithm. Further validation is necessary for this new algorithm before adopting this method.
- Publication
Clinical Laboratory Science, 2017, Vol 31, Issue 3, p163
- ISSN
0894-959X
- Publication type
Academic Journal