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 i...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0091824&type=printable |
| _version_ | 1826799306070294528 |
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| author | Dong Chen Zhao Yang Han Xia Jun-Fu Huang Yang Zhang Tian-Nun Jiang Gui-Yu Wang Zheng-Ran Chuai Wei-Ling Fu Qing Huang |
| author_facet | Dong Chen Zhao Yang Han Xia Jun-Fu Huang Yang Zhang Tian-Nun Jiang Gui-Yu Wang Zheng-Ran Chuai Wei-Ling Fu Qing Huang |
| author_sort | Dong Chen |
| collection | DOAJ |
| description | 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. |
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| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
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| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj.art-e79cb035336b43cfbb5ed79642eb7b5e2025-02-21T05:34:53ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0194e9182410.1371/journal.pone.0091824Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.Dong ChenZhao YangHan XiaJun-Fu HuangYang ZhangTian-Nun JiangGui-Yu WangZheng-Ran ChuaiWei-Ling FuQing HuangGenotyping 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0091824&type=printable |
| spellingShingle | Dong Chen Zhao Yang Han Xia Jun-Fu Huang Yang Zhang Tian-Nun Jiang Gui-Yu Wang Zheng-Ran Chuai Wei-Ling Fu Qing Huang Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. PLoS ONE |
| title | Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. |
| title_full | Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. |
| title_fullStr | Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. |
| title_full_unstemmed | Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. |
| title_short | Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. |
| title_sort | enhanced specificity of tpmt 2 genotyping using unidirectional wild type and mutant allele specific scorpion primers in a single tube |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0091824&type=printable |
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