2011年9月
A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation
Analytical Biochemistry
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- 巻
- 416
- 号
- 2
- 開始ページ
- 211
- 終了ページ
- 217
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.ab.2011.05.017
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
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, (Ill) 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. (C) 2011 Elsevier Inc. All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/j.ab.2011.05.017
- CiNii Articles
- http://ci.nii.ac.jp/naid/80021902441
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/21669180
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000293046400012&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1016/j.ab.2011.05.017
- ISSN : 0003-2697
- CiNii Articles ID : 80021902441
- PubMed ID : 21669180
- Web of Science ID : WOS:000293046400012