建立检测细胞色素P450酶与紫杉醇代谢相关突变位点的基因芯片分型方法

摘要

目的 建立针对与临床抗癌药物紫杉醇代谢相关的细胞色素P450(CYP450)酶基因多态性位点的快速、准确、高通量的基因芯片基因分型方法.方法 选取CYP450酶2C8*3、3A4* 18、3A5*3C等3个与紫杉醇代谢相关突变位点,根据基因库中报道的序列,设计每个基因多态性位点的野生型和突变型探针,在突变位点2侧设计PCR扩增引物,PCR片段长度应＜200 bp,并构建标准质粒.探针在3'端氨基修饰,下游引物标记荧光素Cy3,将探针按一定顺序点样于经醛基化处理的玻片制备成基因芯片.人体血液DNA样本分别通过3对引物扩增后与基因芯片上的探针杂交,通过扫描图像和配套软件对结果进行分析和判断.同时,对50份血液样本进行检测.结果 通过标准质粒杂交结果显示,每个位点对应的1对探针均能将野生型和突变型质粒进行准确地区分,无非特异性杂交信号出现;检测50份血液样本,CYP2C8 * 3位点的突变率为2%,CYP3A4 * 18位点均为野生型,CYP3A5 * 3C位点的突变率为62%.同时,通过测序法进行验证,基因芯片方法与测序方法的结果完全一致.结论 建立的同时检测抗癌药物紫杉醇代谢相关的CYP450酶基因多态性位点CYP2C8*3、CYP3A4 * 18、CYP3A5 * 3C的基因芯片分型方法快速、准确,结果可靠,重复性好,可用于指导紫杉醇患者的个体化用药,并为分析个体患者对于紫杉醇药物体内代谢提供了一个高通量技术平台.%Objective To develop an accurate,rapid,high throughout genotyping method based on oligonucleotide microarray for cytochrome P450 gene polymorphisms related to paclitaxel metabolism.Methods The mutant points of 2C8 * 3,3A4 * 18 and 3A5 * 3C from cytochrome P450 gene were regarded as targets.Based on the sequences in the GenBank,the wild-type and mutant-type probes were specially designed for each mutant point.PCR primers were located in the both sides of mutant point,and furthermore the fragments of PCR products were less than 200 bp.Each type of standard plasmids was constructed.Thus,all the olignucleotide probes were modified with 3'amino-group,and the reverse primers were labeled with fluorescein (Cy3).The probes were immobilized onto certain glass slides.The specific fragments of three genes were amplified and then hybridized with oligonucleotide microarray.The results were analyzed by using certain software.Finally this assay was applied to detect 50 clinical blood specimens.Results When PCR products from standard plasmids were hybridized with DNA microarray,the corresponding probes produced positive signals.Meanwhile,the non-specific hybridization signals did not appear.The results of clinical specimens showed that the mutant rate of CYP2C8 * 3 was 2%.The point of CYP3A4 * 18 for all the clinical specimens was wild-type and the mutant rate of CYP3A5 * 3C was 62%. Meanwhile,the results from detecting 50 clinical blood specimens using oligonucleotide microarray were the same as sequencing analysis.Conclusions Oligonucleotide microarray is a reliable and accurate genotyping assay for cytochrome P450 2C8 * 3.3A4 * 18 and 3A5 * 3C polymorphisms related to paclitaxel simultaneously.This genotyping assay is a high-throughout method for guiding personalized therapy and analyzing metabolism of paclitaxel in vivo.