Enhanced specificity of TPMT2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube

• Published in: PloS one 2014-04, Vol.9 (4), p.e91824-e91824
• Main Authors: Chen, Dong, Yang, Zhao, Xia, Han, Huang, Jun-Fu, Zhang, Yang, Jiang, Tian-Nun, Wang, Gui-Yu, Chuai, Zheng-Ran, Fu, Wei-Ling, Huang, Qing
• Format: Article
• Language: English
• Subjects: Adverse and side effects; Alleles; Allelomorphism; Analysis; Base Sequence; Biology and Life Sciences; Deoxyribonucleic acid; DNA; DNA Mutational Analysis - methods; DNA Mutational Analysis - standards; DNA polymerase; DNA Primers - genetics; Drugs; Experiments; Fluorescence; Genotype; Genotyping; Genotyping Techniques - methods; Genotyping Techniques - standards; Hospitals; Humans; Industrial design; Laboratories; Medical research; Medicine; Medicine and Health Sciences; Methyltransferase; Methyltransferases - genetics; Mutation; Oligonucleotides - chemistry; Oligonucleotides - genetics; Optimization; Polymorphism, Single Nucleotide; Primers; Quality Control; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Research and Analysis Methods; Sensitivity and Specificity; Signal-To-Noise Ratio; Substrate Specificity; Taguchi methods; Thiopurine S-methyltransferase
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0091824

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.