Methylation preserving polymerase chain reaction

概述：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的CpG二核苷酸中，在基因的启动子区很常见。启动子区域甲基化状态的改变会导致该基因表达的改变。甲基化状态的缺陷与许多疾病有关。分析5-mC的常用方法需要数千个细胞。需要提高灵敏度的方法。我们建议开发保留甲基化的聚合酶链反应（mpPCR）作为一种工具来扩增少量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，将样本加热使DNA变性。其次，引物退火到模板。第三步，利用DNA聚合酶延长引物，复制模板。这一步产生了半甲基化DNA，其中原始链被甲基化，新合成的链未甲基化。在第四步，使用人甲基转移酶Dnmt1将半甲基化的DNA转化为完全甲基化的DNA。产物的数量随着每次循环呈指数增长，并且产物可以使用任何传统的甲基胞嘧啶分析技术进行分析。

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.