1. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube
- Author
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Weiling Fu, Han Xia, Dong Chen, Tian-Nun Jiang, Gui-Yu Wang, Jun-Fu Huang, Qing Huang, Zhao Yang, Zheng-Ran Chuai, and Yang Zhang
- Subjects
Genetic Screens ,Genotyping Techniques ,DNA Mutational Analysis ,Gene Identification and Analysis ,Oligonucleotides ,lcsh:Medicine ,Gene Expression ,Signal-To-Noise Ratio ,Pathology and Laboratory Medicine ,law.invention ,Substrate Specificity ,law ,Genotype ,Molecular Cell Biology ,Medicine and Health Sciences ,lcsh:Science ,Polymerase chain reaction ,Genetics ,Clinical Chemistry ,Multidisciplinary ,Thiopurine methyltransferase ,Genomics ,Clinical Laboratory Sciences ,Bioassays and Physiological Analysis ,Transcriptome Analysis ,Research Article ,Quality Control ,Clinical Pathology ,Biology ,Research and Analysis Methods ,Real-Time Polymerase Chain Reaction ,Polymorphism, Single Nucleotide ,Sensitivity and Specificity ,Engineering optimization ,Molecular Genetics ,Taguchi methods ,Diagnostic Medicine ,Humans ,Genotyping ,Alleles ,DNA Primers ,Base Sequence ,lcsh:R ,Wild type ,Biology and Life Sciences ,Computational Biology ,Cell Biology ,Methyltransferases ,Genome Analysis ,biology.protein ,lcsh:Q ,Orthogonal array ,Genome Expression Analysis ,Biochemical Analysis - Abstract
Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.
- Published
- 2014