芯片技术用于APC基因启动子甲基化定量检测  

Analysis of methylation profiles of APC gene promoter in lung cancer by methylation-specific microarray

作　　者：王贤[1] 潘世扬[1] 陆祖宏[2] 程璐[2] 魏源华[1] 张丽霞[1] 陈丹[1] 张寄南[1] 

机构地区：[1]南京医科大学第一附属医院,南京210029 [2]东南大学生物科学与医学工程系吴健雄实验室

出　　处：《临床检验杂志》2006年第3期199-202,共4页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省政府"135"重点实验室科研基金(SK200205);诊断心肌疾病的生物芯片研制与应用(BG2003033)。

摘　　要：目的研制抑癌基因APC启动子1A的甲基化定量检测芯片,对临床标本进行初步定量检测分析。方法采用脐带血DNA克隆体作为阴性、阳性质控品建立芯片,提取6例健康人的肺组织DNA、6例肺癌患者的肺组织和癌组织DNA,进行亚硫酸盐化学修饰,以甲基化特异性引物进行基因扩增,与探针杂交检测,并进行甲基化特异性序列分析。结果甲基化阳性、阴性质控品的芯片检测与测序结果吻合。687、707、714、719和726共5个位点的荧光强度标准曲线的R2为0．93-0．99。687、707、714、719和726共5个位点非甲基化的检测范围:(0±8．7)％、(0±17．6)％、(0±17)％、(0±13)％、(0±8)％;甲基化杂合型的检测范围:(50±3．6)％、(50±6．9)％、(50±3．5)％、(50±8．5)％、(50±7．3)％。标本的芯片检测与测序结果一致。结论本研究首次建立了以微阵列技术为基础的APC基因启动子甲基化定量检测芯片。Objective To determine methylation profiles and heterozygosis status of APC gene promotor in lung cancer by using newly developed methylation-specific microarray（MSM）.Method A methylation-specific microarray was devoleped with cord blood DNA methylation positive and negative control.We detected 2 normal PBL,8normal lung tissues,18 lung cancer tissues and corresponding 18pericancerous lung tissues .Bisulfite-treated DNA was used as template for PCR amplification.The PCR product was then hybridized to a set of oligonuleotide arrays where methylated and unmethylated cytosine at specific nucleotide positions was discriminated,and quantitative differences of hybridization were determined by fluorescence analysis.Results Methylation negative and positive pattems of APC gene promoter 1A were mapped and the results were validated by bisulphite DNA sequencing,The standard curves for fluorescence intensity from 5 CpC sites were mapped to deteet hypermethylation quantitatively（R^2=0.93-0.99）.The range of methylated ratio was defined to detect the unmethylated cytosine（M0：0%±8.7%,M1：0%±17.6%,M2：0%±17%,M3：0%±13%,M4：0%±8%）.The range of methylated ratio of heterozygosis was defined for detection （M0：50%±3.6%,M1：50%±6.9%,M2：50%±3.5%,M3：50%±8.5%,M4：50%±7.3%）.The results of hybridization on microarray were parallel to bisulfite DNA sequencing.The heterozygosis was only found in pericancerous and carcinomatous lung tissues .The frequencies of methylation of CpC site M0,M1,M2,M3 and M4 in carcinomatous lung tissues were 68%,83%,78%,56%,68%,The methylation levels in pericancerous lung tissues were：50%（M0）,68%（M1）,68%（M2）,44%（M3）,61%（M4）.The frequencies of methylation of CpC site M0,M1,M2,M3,and M4 in normal lung and PBL were 30%,30%,10%,10%.Conclusion Methylation pattern and methylated heterozygosis sstatus of APC gene in lung cancer can be easy approved with microarray technique.

关 键 词：APC基因 甲基化 芯片 定量 

分 类 号：Q789[生物学—分子生物学]