Methylation preserving polymerase chain reaction

描述(申请人提供)：摘要。5-甲基胞嘧啶(5-MC)是一种与表观遗传相关的修饰核苷酸。5-MC通常存在于真核生物DNA中的CpG二核苷酸中，普遍存在于基因的启动子区域。启动子区域甲基化状态的变化会导致该基因表达的变化。甲基化状态的缺陷与一系列疾病有关。常用的5-MC分析方法需要数千个细胞。需要具有更高灵敏度的方法。我们建议发展甲基化保护聚合酶链式反应(MpPCR)作为一种工具，在保持起始材料的甲基化状态的同时扩增少量DNA。MpPCR的每个周期由四个步骤组成。首先，将样本加热以变性DNA。其次，将引物与模板进行退火热处理。第三，DNA聚合酶被用来延伸引物，复制模板。这一步骤产生了半甲基化的DNA，其中原始的链是甲基化的，而新合成的链是去甲基化的。在第四步中，人类甲基转移酶Dnmt1被用来将半甲基化的DNA转化为完全甲基化的DNA。产物的量随着每个周期呈指数增长，并且可以使用任何传统的甲基胞嘧啶分析技术来分析产物。 公共卫生相关性：基因启动子区域甲基化状态的缺陷与包括癌症在内的许多疾病有关。目前的甲基化分析技术需要相对大量的样品。我们建议开发一种能够分析小组织和单个细胞的技术。

DESCRIPTION (provided by applicant): Summary. 5-methyl cytosine (5-mC) is a modified nucleotide that is associated with epigentic inheritance. 5-mC is usually found in CpG dinucleotides in eukaryotic DNA, which are common in the promoter region of genes. Changes in the methylation status of a promoter region results in changes in the expression of that gene. Defects in the methylation status have been associated with a wide range of diseases. Common methods for the analysis of 5-mC require thousands of cells. There is a need for methods with improved sensitivity. We propose to develop methylation preserving polymerase chain reaction (mpPCR) as a tool to amplify small amounts of DNA while preserving the methylation status of the starting material. Each cycle of mpPCR consists of four steps. First, a sample is heated to denature the DNA. Second, primers are annealed to the template. Third, DNA polymerase is used to extend the primers, copying the template. This step creates hemi-methylated DNA, where the original strand is methyated and the newly synthesized strand is unmethylated. In the fourth step, the human methyl transferase Dnmt1 is used to convert hemi-methylated DNA to fully methylated DNA. The amount of product increases exponentially with each cycle, and the product may be analyzed using any conventional methylcytosine analysis technology. PUBLIC HEALTH RELEVANCE: Defects in the methylation status of the promoter region of a gene are associated with many diseases, including cancer. Current technologies for methyation analysis require relatively large amounts of sample. We propose to develop a technology that allows analysis of small tissues and single cells.