摘要
目的建立一种基于错配杂交化学发光检测p16基因启动子区过甲基化的定量分析方法.方法用亚硫酸氢钠修饰基因组DNA,所有未甲基化的胞嘧啶都被转变为尿嘧啶,而甲基化的胞嘧啶则不发生变化.设计合成1对不含CpG位点的引物,同时扩增甲基化或非甲基化目的DNA片段,用2条分别与甲基化及非甲基化CpG位点互补的寡核苷酸探针与扩增产物进行杂交及化学发光检测,通过探针杂交信号强度比确定样品DNA中甲基化的p16基因的比例.结果错配杂交化学发光法具有较好的精密度(CV=5.2%～6.4%)和灵敏度(2.5×10-4pmol),检测已知混合样品的p16基因甲基化率分别为0%、23%、52 %、70% 及93%,与实际结果一致.结论本研究建立的错配杂交化学发光法是一种检测快速、操作简便的p16基因甲基化的定量检测方法.
Objectives To establish a novel method for quantitative detection of p16 promoter hypermethylation by use of mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite, so that all unmethylated cytosines were converted to uracil and a pair of primer, which had no CpG sites, was designed for amplification target DNA contained methylated or unmethylated CpG sites. Two oligonucleotide probes, which perfectly match the methylated and unmethylated CpG sequences respectively, were synthesized. The PCR products spanning CpG sites were hybridized with two oligonucleotide probes, aThe hybrids were detected by chemiluminescence. The percentage of methylated target sequences could be estimated by calculating the ratio of signals of two probes. Results The method showed an excellent precision（ CV = 5.2% - 6.4% ） and detection limit （2. 5 × 10^-4 pmol）. The mixtures sampls of methylated and unmethylated DNA were analysed by this method and the results were agreed with the target value. Conclusion Compared with the existing methods, this assay is a simple, sensitive and specific method for quantitative analysis of the promoter methylation of p16 gene.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2005年第10期1057-1059,共3页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金资助项目(39990570)
关键词
P16基因
甲基化
错配杂交化学发光法
p16 gene
Methylation
Chemihuminescence quanititative detection
