title: A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.
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.
authors.affiliation: Department of Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu-cho, Hokkaido 061-0293, Japan. arakawa@hoku-iryo-u.ac.jp
authors.forename: Toshiya
authors.initials: T
authors.lastname: Arakawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohta
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Abiko
authors.forename: Miki
authors.initials: M
authors.lastname: Okayama
authors.forename: Itaru
authors.initials: I
authors.lastname: Mizoguchi
authors.forename: Taishin
authors.initials: T
authors.lastname: Takuma
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D018899:CpG Islands; D051546:Cyclooxygenase 2 / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D049452:Green Fluorescent Proteins; D051546:Cyclooxygenase 2; C045073:Cre recombinase; D019426:Integrases