APC基因甲基化定量检测芯片的建立

A methylation-specific oligonuceotide microarray for quantitative analysis of APC gene

目的建立抑癌基因APC(adenomatous polyposis coli,APC)启动子1A的甲基化定量芯片检测方法。方法选取一段420bp的APC基因启动子1A CpG密集序列作为靶序列,针对M0、M1、M2、M3、M45个CpG靶位点,设计一套检测甲基化与非甲基化的探针。采用脐带血DNA克隆体作为阴性、阳性质控品。结果甲基化阳性、阴性质控的芯片结果与测序吻合。每组探针中荧光强度由强至弱依次为,阳性质控(甲基化):探针1>2、3>4;阴性质控(非甲基化):探针3>4、1>2。5个位点的5条荧光强度标准曲线,R2范围是0.93～0.99。M0、M1、M2、M3、M45个位点甲基化杂合型的检测范围分别为50.0%±3.6%、50.0%±6.9%、50.0%±3.5%、50.0%±8.5%、50.0%±7.3%。结论建立了APC基因启动子5个CpG位点的甲基化定量检测芯片。

Objective To develop a methylation-specific oligonucleotide microarray for analyzing APC （adenomatous polyrosis coli） gene promotor methylation pattern. Methods A 420 bp segment of the APC gene promoter 1A was selected as the target sequence including the most densely packed CpG site of the islands. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of those CpG sites M0, M1, M2, M3, M4. A test sample was hybridized to a set of oligonucleotide arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions,and quantitative differences in hybridization were determined by fluorescence analysis. Results Methylation negative and positive patterns of APC gene promoter 1A were mapped and the results were validated by bisulphite DNA sequencing.Standardization curves for fluorescence intensity were mapped to quantitatively detect hypermethylation （R^2 = 0.93 - 0.99）. The range of the methylated ratio of the heterozygosis was defined to detect heterozygosis （M0 50.0 %± 3.6 %, M1 50.0 % ± 6.9 %, M2 50.0 % ± 3.5 %, M3 50.0 %± 8.5 %, M4 50.0 %± 7.3 % ）. The results of microarray hybridization were in agreement with bisulfite DNA sequencing. Conclusion The methylation oligonucleotide microarray can be applied as a useful and powerful tool to perform large quantitative detection of methylation patterns.