基因芯片检测肿瘤病人多药耐药基因1多态性方法的建立  

Establishment of genotyping method for multi-drug resistance gene 1 polymorphisms with DNA microarray

作　　者：徐笑红[1] 林莉[2] 束新华[1] 包叶江[1] 刘旗[1] 金大智[3] 余新民[1] 

机构地区：[1]浙江省肿瘤医院,杭州310022 [2]军事医学科学院附属307医院,北京100071 [3]浙江省疾病预防控制中心,杭州310051

出　　处：《中国卫生检验杂志》2009年第4期874-877,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省医药卫生科学研究基金资助项目(2004A011)

摘　　要：目的:多药耐药基因1(MDR1)多态性检测是临床预测肿瘤患者化疗疗效的有效方法。利用基因芯片技术,建立快速、准确、高通量检测临床肿瘤病人多药耐药基因1多态性位点的分型方法。方法:分别选取多药耐药基因1中外显子2上的C4T和A61G、外显子11上的G1199A、外显子12上的C1236T、外显子21上的C2650T和G2677T、外显子26上的T3421A和C3435T共8个基因位点,根据GenBank报道的序列,针对每个基因多态性位点设计野生型、突变型探针及对应PCR扩增引物,构建每个多态性位点标准质粒。探针在3′端氨基修饰,按顺序制备基因芯片。下游引物标记荧光素Cy3,血液DNA样本分别通过两对两重不对称PCR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,采用该方法检测40例临床样本。结果:通过标准质粒杂交结果显示,8个位点的探针均能准确地区分野生型和突变型质粒,无非特异性杂交信号;检测40例临床样本,结果显示样本中-4CC,62AA,1199GG,2650CC,3420TT位点未见突变,而发生在1236CC,2677TT,3435CC位点的突变频率较高。1236CC的突变频率为35%,2677GG的突变频率为32.5%,而3435CC的突变频率为30%,此外2677TT和3435TT具有连锁关系,在8例2677TT的患者中有6例也是3435TT基因型。同时,通过测序法进行验证,结果显示基因芯片方法与测序方法的结果完全一致。结论:本研究所建立的基因芯片方法可同时对8个多药耐药基因1多态性位点进行分型,该方法快速、准确,结果可靠,重复性好,为临床上预测肿瘤患者对化疗的疗效提供一种可行的检测手段。Objective： The detection of multi - drug resistance gene 1 polymorphisms is an effective assay for predicting thera- peutic effect. An accurate, rapid, high throughout method ideveloped for genotyping multi - drug resistance gene 1 polymorphisms based on oligonucleotide microarray. Methods：8 mutant points of C4T, A61G in exon 2, G1199A in exon 11, C1236T in exon12, C2650T, G2677T in exon 21, T3421A, C3435T in exon 26 from multi - drug resistance gene 1 were regarded as targets. Based on the sequences in the GenBank, the wild - type, mutant - type probes and PCR primers according to each mutant point were designed. Standard plasmids was constructed. Then all the olignucleotide probes modified with 3 amino - group were immobilized onto glass slides. Meanwhile, the reverse primers were labeled with fluorescein （ Cy3 ）. Genomic DNA was amplified by using two asymmetric duplex - PCR reactions, and then hybridized with DNA microarray. The results were analyzed by using software. In final the assay reported here was applied to detect 40 clinical specimens. Results：The results indicated that when PCR products from standard plasmids were hybridized with DNA microarray, the corresponding probes produced positive signals. Furthermore, the non - specific hybridization signals did not appear. The results of clinical specimens showed that the point of -4CC, 62AA, 1199GG, 2650CC, 3420TF for all the clinical specimens was wild - type, and the mutant rate of 1236CC was 35%, the mutant rate of 2677GG was 32. 5%, the mutant rate of 3435CC was 30%. In addition, 2677TT and 3435TT had a linkage relationship, there were 6 mutant points of C3435T in 8 mutant points appeared in G2677T. Meanwhile, the above results from detecting 40 clinical specimens using DNA microarray were the same to that of the direct sequencing method. Conclusion： DNA mieroarray described in this paper is a reliable and accurate genotyping assay for 8 mutant points of CA-T, A61G in exon 2, G1199A in exon 11, C1236T in exonl2, C2650T, G2677T in exon 21,

关 键 词：多药耐药基因1 多态性 基因芯片 分型 

分 类 号：R446.6[医药卫生—诊断学]