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 language | English |
|---|---|
| Pages (from-to) | 846-854 |
| Number of pages | 9 |
| Journal | BioTechniques |
| Volume | 36 |
| Issue number | 5 |
| Publication status | Published - May 2004 |
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ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
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 journal › Article
}
TY - JOUR
T1 - Methylation assay by nucleotide incorporation
T2 - A quantitative assay for regional CpG methylation density
AU - Yamamoto, Takeshi
AU - Nagasaka, Takeshi
AU - Notohara, Kenji
AU - Sasamoto, Hiromi
AU - Murakami, Jun
AU - Tanaka, Noriaki
AU - Matsubara, Nagahide
PY - 2004/5
Y1 - 2004/5
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=2442554066&partnerID=8YFLogxK
M3 - Article
C2 - 15152605
AN - SCOPUS:2442554066
VL - 36
SP - 846
EP - 854
JO - BioTechniques
JF - BioTechniques
SN - 0736-6205
IS - 5
ER -