目的 建立葡萄糖醛酸转移酶1A3(UDP-glucuronosyltransferase,UGT1A3)一号外显子上-31T/C、-133C/T及-140T/C 3个位点基因型的检测方法,考察这3个位点的基因多态性在中国健康人群中的发生频率。方法 采用两步等位基因特异扩增法,设计葡萄糖醛酸转移酶1A3一号外显子的引物扩增基因组DNA,在此基础上针对每一个位点设立两个分别含有野生型或突变型引物的平行反应管,各包含一条3′端最后一位碱基分别与野生型或突变型配对的上游引物及一条相同的下游引物来扩增目的基因,采用1%琼脂糖凝胶电泳考察不同引物的扩增情况,以区分位点基因型,并抽样进行直接焦磷酸测序以验证方法的准确性。结果 123例(男89例,女34例)无亲缘关系的健康受试者中-31位点等位基因T和C的频率分别为0.776和0.224,-133位点等位基因C和T的频率分别为0.935和0.065,-140位点等位基因T和C的频率分别为0.915和0.085。UGT1A3*1/*1、UGT1A3*1/*2、UGT1A3*2/*2、UGT1A3*1/*4及UGT1A3*4/*4携带者分别有59名、17名、1名、12名及1名。结论 中国健康人群中葡萄糖醛酸转移酶1A3一号外显子-31、-133及-140位点存在遗传多态性;本试验建立的基因型测定与分析方法特异、准确,可满足UGT1A3遗传多态性研究的需要。
Abstract
Objective To establish a method to determine the genotypes of the three SNPs(-31T/C, -133C/T and -140T/C) of uridinediphosphateglucuronosyltransferase 1A3(UGT1A3)and to preliminarily analyze the genetic frequencies of different genotypes among healthy Chinese population.Methods The genotypes of the three SNPs(-31T/C, -133C/T and -140T/C) of 123 healthy Chinese subjects(89 males, 34 females ) were screened by two-step allele specific amplification(two-step ASA) method. Firstly, a pair of primers of the first exon for the amplification of the genomic DNA were designed. And then two parallel reaction tubes were set up for each site which included a forward primer whose last base of 3′end pair with wild type or mutant type and an identical reverse prime for the amplification of the target gene.The amplification Results were examined to differentiate the genotypes. The validity was verified by direct sequencing.Results Among the 123 subjects, the frequencies of -31T and -31C were 0.776 and 0.224, of -133C and -133T were 0.935 and 0.065, of -140T and -140C were 0.915 and 0.085, respectively. Based on the genotyping Results, there were 59 cases of UGT1A3*1/*1, 1 case of UGT1A3*2/*2,17 cases of UGT1A3*1/*2, 12 cases of UGT1A3*1/*4, and 1 case of UGT1A3*4/*4.Conclusion There are genetic polymorphisms of UGT1A3 in healthy Chinese population and the mutation frequency is remarkable. The gene detection method established in this study is verified to be sensitive and specific.
关键词
葡萄糖醛酸转移酶1A3 /
基因多态性 /
两步等位基因特异扩增法
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Key words
UDP-glucuronosyltransferase 1A3 /
polymorphisms /
two-step ASA
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中图分类号：
R915.2
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