| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 95 === Mammalian DNA methylation is an epigenetic process involving in transcriptional gene silencing, suppression of transposable elements and chromatin structure modulation. Aberrant methylation of CpG-rich promoter regions has been found in many human cancers. Methylation profiling also poses an important topic for study of embryonic stem cell and somatic cell cloning. The current method for methylation status analysis relies on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty for acquiring a methylation profiling is often limited amount of genomic DNA obtained and complex procedures of bisulfite treatment dramatically reduces the effective template for PCR analysis. In this report, we developed degenerated oligonucleotide primer (DOP)-PCR to pre-amplify bisulfite-modified genomic DNA for increasing experiment materials for further methylation profiling analysis. A DOP pair for genome-wide amplification were designed: 1st 3’ DOP, CTCGAGCTGHHHHHAACTAC where H is a mixture of base consisting 50% A, 25% T, and 25% C and 2nd 5’ DOP, CTCGAGCTGDDDDDGTTTAG where D is a mixture of base consisting 50% T, 25% G, and 25% A. Bisulfite-modified genomic DNA from cell line, cord blood cell or cells obtained by laser capture micro-dissection was amplified to thousand folds by our method. Eight cancer-related genes were randomly selected for subsequent MSP analysis. It was found that methylation status of these genes remained identical to that derived from the original unamplified template.
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