TY - JOUR
T1 - A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation
AU - Arakawa, Toshiya
AU - Ohta, Tohru
AU - Abiko, Yoshihiro
AU - Okayama, Miki
AU - Mizoguchi, Itaru
AU - Takuma, Taishin
N1 - Funding Information:
This work was supported by MEXT.HAITEKU ( 2007 ). We acknowledge W.L. Smith (University of Michigan) for providing valuable comments and advice on the manuscript and D.L. Dewitt (Michigan State University) for kindly providing mouse COX-2 promoter vector. We also thank T. Inoue (Qiagen) for providing technical support of HRM analysis, Hokkaido System Science for providing methylated primers, R Development Core Team (R Foundation for Statistical Computing) for providing R: A Language and Environment for Statistical Computing, and Roger Abey (Eisai) for critical reading of the manuscript.
PY - 2011/9/15
Y1 - 2011/9/15
N2 - 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.
AB - 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.
KW - DNA methylation
KW - Site-specific
KW - Transcriptional regulation
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U2 - 10.1016/j.ab.2011.05.017
DO - 10.1016/j.ab.2011.05.017
M3 - Article
C2 - 21669180
AN - SCOPUS:80155147107
VL - 416
SP - 211
EP - 217
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -
