Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.
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.
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Dong
D
Chen
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Zhao
Z
Yang
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Han
H
Xia
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Jun-Fu
JF
Huang
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Yang
Y
Zhang
Department of Blood Transfusion, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Tian-Nun
TN
Jiang
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Gui-Yu
GY
Wang
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Zheng-Ran
ZR
Chuai
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Wei-Ling
WL
Fu
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China.
Qing
Q
Huang
D000483:Alleles; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards; D017931:DNA Primers / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods* / Q000592:standards; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D059629:Signal-To-Noise Ratio; D013379:Substrate Specificity
D017931:DNA Primers; D009841:Oligonucleotides; D008780:Methyltransferases; C022745:thiopurine methyltransferase