High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes
High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes
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DOI：
10.1136/mp.52.5.295
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发表时间：
1999-10-01
期刊：
影响因子：
--
通讯作者：
de la Iglesia, FA
中科院分区：
文献类型：
--
作者：
Shi, MM;Myrand, SP;de la Iglesia, FA
AIMS--苯二酚NAD(P)H氧化还原酶(NQO1)催化对苯二酚的双电子还原反应，在对苯二酚及类似活性化合物的活化和解毒过程中起着重要作用。外显子6的单核苷酸多态导致第187位密码子的氨基酸由脯氨酸变为丝氨酸。这种变异等位基因与NQO1酶活性降低和癌症风险增加有关。本研究的目的是建立一种快速的基因分型方法，用于流行病学和临床研究与这种基因多态相关的潜在生物学和毒理学影响。方法-建立了一种高通量的荧光探针基因分型方法来筛选这种单核苷酸多态。本试验利用Tag聚合酶的5‘核酸酶活性与荧光TaqMan探针相结合。用限制性片段长度多态法和直接测序法对TaqMan基因分型方法进行了验证。结果：该方法可用于大规模人群中已知基因多态性的快速筛查。在143名无关个体中，Pro/Pro(野生型)、Pro/Ser(杂合型)和Ser/Ser(突变型)基因型分别为69.2%、26.6%和4.2%。结论：该基因分型方法具有较高的准确性，可用于自动化大规模基因分型研究。
Aims-The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism.Methods-A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Tag polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing.Results-This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively.Conclusions-This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.