Methylation preserving polymerase chain reaction

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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.