Methylation preserving polymerase chain reaction

描述（由申请人提供）：摘要。5-甲基胞嘧啶（5-mC）是与表观遗传相关的修饰的核苷酸。5-mC通常存在于真核生物DNA中的CpG二核苷酸中，常见于基因的启动子区。启动子区域甲基化状态的变化导致该基因表达的变化。甲基化状态的缺陷与多种疾病有关。用于分析5-mC的常用方法需要数千个细胞。需要具有改进的灵敏度的方法。我们建议开发甲基化保留聚合酶链反应（mpPCR）作为一种工具，以扩增少量的DNA，同时保留起始材料的甲基化状态。mpPCR的每个循环由四个步骤组成。首先，加热样品以使DNA变性。第二，引物与模板退火。第三，DNA聚合酶用于延伸引物，复制模板。这一步产生半甲基化的DNA，其中原始链被甲基化，新合成的链未甲基化。在第四步中，使用人甲基转移酶Dnmt 1将半甲基化DNA转化为完全甲基化DNA。产物的量随着每个循环呈指数增加，并且可以使用任何常规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许多疾病相关，包括癌症。目前的甲基化分析技术需要相对大量的样品。我们建议开发一种可以分析小组织和单细胞的技术。

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.