A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation

Toshiya Arakawa, Tohru Ohta, Yoshihiro Abiko, Miki Okayama, Itaru Mizoguchi, Taishin Takuma

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)211-217
Number of pages7
JournalAnalytical Biochemistry
Issue number2
Publication statusPublished - 2011 Sep 15
Externally publishedYes


  • DNA methylation
  • Site-specific
  • Transcriptional regulation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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