Methylation preserving polymerase chain reaction

甲基化保留聚合酶链反应

• 批准号: 7976428
• 负责人: Norman J Dovichi
• 金额: $ 21.25万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976428
• 关键词: Biological; Biological Preservation; Calibration; Cells; CpG dinucleotide; Cytosine; DNA; DNA-Directed DNA Polymerase; Deamination; Defect; Detection; Development; Disease; Epigenetic Process; Gene Expression; Genes; Heating; Human; Malignant Neoplasms; Methods; Methylation; Methyltransferase; Nucleotides; Pattern; Polymerase; Polymerase Chain Reaction; Promoter Regions; Sampling; Site; Technology; Temperature; Tissues; Transferase; improved; methyl group; public health relevance; tool

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.

描述（申请人提供）：摘要。 5-甲基胞嘧啶（5-MC）是一种与表观遗传有关的修饰核苷酸。 5-MC通常在真核DNA中的CpG二核苷酸中发现，这在基因的启动子区域中很常见。启动子区域的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。分析5-MC的常见方法需要数千个细胞。需要提高灵敏度的方法。我们建议开发甲基化保存聚合酶链反应（MPPCR）作为扩增少量DNA的工具，同时保留起始材料的甲基化状态。 MPPCR的每个周期都由四个步骤组成。首先，将样品加热以使DNA结染。其次，将引物退火到模板。第三，DNA聚合酶用于扩展引物，复制模板。此步骤产生了半甲基化的DNA，其中原始链被甲化化，而新合成的链未被甲基化。在第四步中，人甲基转移酶DNMT1用于将半甲基化的DNA转化为完全甲基化的DNA。产品的量随着每个周期的形式呈指数增长，并且可以使用任何常规的甲基环肽分析技术对产品进行分析。 公共卫生相关性：基因启动子区域的甲基化状况缺陷与包括癌症在内的许多疾病有关。当前用于甲基化分析的技术需要相对较大的样本。我们建议开发一种允许分析小组织和单细胞的技术。