DHPLC方法对中国人iNOS基因Tsp多态性的发现与初步证实

目的: 探讨中国人iNOS基因是否存在单核苷酸多态性及其在中国人中的频率.方法:DHPLC方法结合直接测序和PCR-RF LP技术,在有代表性的中国人中进行筛查.结果:在iNOS基因全部被测外显子中,有6 个外显子发现可疑双峰图型,可能存在杂合突变,对其测序后在iNOS基因第16外显子发现C→T突变 (称为Tsp多态性),使编码的氨基酸由丝氨酸 (serine,Ser)改变为亮氨酸 (leucine,Leu),设计PCR-RFLP方法并监测全部测序样本,PCR -RFLP与测序结果一致.在有代表性的43例中国人中,Ser和Leu等位基因频率分别为75.58% (65/86)和24.4 2% ( 21/86);三种基因型观察频率分别潍 Ser/Ser:62.79%; Ser/Leu:25.58%; Leu/Leu:1 1.63%, 与期望频率(分别为57.12%, 36.91%和5.96%)差异无显著性(P>0.05),说明符合H ardy -Weinberg遗传平衡定律.结论:中国人群中存在iNOS基因Ts p多态性,且频率较高.

Tsp polymorphism of iNOS gene was found and identified in Chines by denaturing high performance liquid chromatography

To detect the single nucleotide polymo rphi sms (SNPs) in iNOS gene, and its frequency in Chinese population.Method s: Denatur ing high performance liquid chromatography (DHPLC) combined w ith dire ct sequence and PCR-RFLP techniques were used in representative Chines e populat I on.Results:Among all detected exons of iNOS gene, si x exons wer e found to show possible heteroduplex double peaks, which meant pos sible heteroduplex samples. S equencing was conducted among the possible samples , and only exon 16 was found t o have a heterozygous C→T mutation (called Tsp p olymorphiam), which changed the coding amino acid from serine (TCG) to leucine (TTG). PCR-RFLP method was desi g ned to re-identify the result of sequencing. Results indicate d that all sequenci ng identified heterozygous samples were al so detected to be heterozygous using P CR-RFLP method. The allele frequencies f or Ser and Leu were 75.58% (65/86) and 24.42% (21/86) respectively among the 43 Chinese individuals. The o bserved freque ncies of three genotypes in the same population (Ser/Ser, 62.79%; Ser/Leu, 25.58 %; and Leu/Leu, 11.63%) were not dif ferent from those expected from the Hardy-W e inberg equilibrium (57.12%, 36.91 % and 5.96%, respectively; P>0.05); indi cating that they accord with Hardy -Weinberg equilibrium law. Conclusion : Tsp polymorphism exist s in Chinese population, and has a higher frequency.