Methylation assay by nucleotide incorporation: A quantitative assay for regional CpG methylation density

Takeshi Yamamoto, Takeshi Nagasaka, Kenji Notohara, Hiromi Sasamoto, Jun Murakami, Noriaki Tanaka, Nagahide Matsubara

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect 6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.

Original languageEnglish
Pages (from-to)846-854
Number of pages9
JournalBioTechniques
Volume36
Issue number5
Publication statusPublished - May 2004

Fingerprint

Methylation
Assays
Nucleotides
Cytosine
Single-Stranded Conformational Polymorphism
CpG Islands
Uracil
Haptens
DNA
Methyltransferases
Electrophoresis
Polymorphism
Colorectal Neoplasms
Mucous Membrane
Genes
Polymerase Chain Reaction
Neoplasms
hydrogen sulfite

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Yamamoto, T., Nagasaka, T., Notohara, K., Sasamoto, H., Murakami, J., Tanaka, N., & Matsubara, N. (2004). Methylation assay by nucleotide incorporation: A quantitative assay for regional CpG methylation density. BioTechniques, 36(5), 846-854.

Methylation assay by nucleotide incorporation : A quantitative assay for regional CpG methylation density. / Yamamoto, Takeshi; Nagasaka, Takeshi; Notohara, Kenji; Sasamoto, Hiromi; Murakami, Jun; Tanaka, Noriaki; Matsubara, Nagahide.

In: BioTechniques, Vol. 36, No. 5, 05.2004, p. 846-854.

Research output: Contribution to journalArticle

Yamamoto, T, Nagasaka, T, Notohara, K, Sasamoto, H, Murakami, J, Tanaka, N & Matsubara, N 2004, 'Methylation assay by nucleotide incorporation: A quantitative assay for regional CpG methylation density', BioTechniques, vol. 36, no. 5, pp. 846-854.
Yamamoto T, Nagasaka T, Notohara K, Sasamoto H, Murakami J, Tanaka N et al. Methylation assay by nucleotide incorporation: A quantitative assay for regional CpG methylation density. BioTechniques. 2004 May;36(5):846-854.
Yamamoto, Takeshi ; Nagasaka, Takeshi ; Notohara, Kenji ; Sasamoto, Hiromi ; Murakami, Jun ; Tanaka, Noriaki ; Matsubara, Nagahide. / Methylation assay by nucleotide incorporation : A quantitative assay for regional CpG methylation density. In: BioTechniques. 2004 ; Vol. 36, No. 5. pp. 846-854.
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