PMID- 15152605
OWN - NLM
STAT- MEDLINE
DCOM- 20041130
LR  - 20191210
IS  - 0736-6205 (Print)
IS  - 0736-6205 (Linking)
VI  - 36
IP  - 5
DP  - 2004 May
TI  - Methylation assay by nucleotide incorporation: a quantitative assay for regional 
      CpG methylation density.
PG  - 846-50, 852, 854
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 < 1.25% methylated DNA and that calibration curve was 
      linear. With this approach, we determined relative methylation density of 
      O6-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.
FAU - Yamamoto, Takeshi
AU  - Yamamoto T
AD  - International Reagents Corporation, Sysmex Corporation, Kobe, Japan.
FAU - Nagasaka, Takeshi
AU  - Nagasaka T
FAU - Notohara, Kenji
AU  - Notohara K
FAU - Sasamoto, Hiromi
AU  - Sasamoto H
FAU - Murakami, Jun
AU  - Murakami J
FAU - Tanaka, Noriaki
AU  - Tanaka N
FAU - Matsubara, Nagahide
AU  - Matsubara N
LA  - eng
PT  - Comparative Study
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Validation Study
PL  - England
TA  - Biotechniques
JT  - BioTechniques
JID - 8306785
RN  - 0 (Nucleotides)
SB  - IM
MH  - Chromosome Mapping/*methods
MH  - Colorectal Neoplasms/*genetics
MH  - CpG Islands/*genetics
MH  - *DNA Methylation
MH  - Genetic Testing/*methods
MH  - Humans
MH  - Nucleotides/*genetics
MH  - Sequence Analysis, DNA/*methods
MH  - Tumor Cells, Cultured
EDAT- 2004/05/22 05:00
MHDA- 2004/12/16 09:00
CRDT- 2004/05/22 05:00
PHST- 2004/05/22 05:00 [pubmed]
PHST- 2004/12/16 09:00 [medline]
PHST- 2004/05/22 05:00 [entrez]
AID - 10.2144/04365RR02 [doi]
PST - ppublish
SO  - Biotechniques. 2004 May;36(5):846-50, 852, 854. doi: 10.2144/04365RR02.