論文 - 詳細
| RRC ID | 67859 |
|---|---|
| 著者 | Arakawa T, Ohta T, Abiko Y, Okayama M, Mizoguchi I, Takuma T. |
| タイトル | A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation. |
| ジャーナル | Anal Biochem |
| Abstract |
DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation. |
| 巻・号 | 416(2) |
| ページ | 211-7 |
| 公開日 | 2011-9-15 |
| DOI | 10.1016/j.ab.2011.05.017 |
| PII | S0003-2697(11)00325-3 |
| PMID | 21669180 |
| MeSH | Animals Cell Line, Tumor CpG Islands Cyclooxygenase 2 / genetics DNA Methylation* DNA Primers / chemistry DNA Primers / metabolism DNA, Single-Stranded / metabolism Green Fluorescent Proteins / genetics Integrases Mice Nucleic Acid Hybridization Plasmids / chemistry* Plasmids / metabolism Polymerase Chain Reaction / methods* Promoter Regions, Genetic |
| IF | 2.877 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 各媒体での言及数の合計 | 0 |
| リソース情報 | |
| ヒト・動物細胞 | COS-7(RCB0539) |