【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。更多还原

【Abstract】 Objective:The detection of multi-drug resistance gene 1 polymorphisms is an effective assay for predicting therapeutic 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,3420TT 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 microarray described in this paper is a reliable and accurate genotyping assay for 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 polymorphisms simultaneously.This assay provided a feasible measure for predicting therapeutic effect in clinic.更多还原