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Not I‐ Mse I methylation‐sensitive amplified fragment length polymorphism for DNA methylation analysis of human cancers
Author(s) -
Yamamoto Fumiichiro,
Yamamoto Miyako,
Soto Jose Luis,
Kojima Eijiro,
Wang Emily N.,
Perucho Manuel,
Sekiya Takao,
Yamanaka Hiroyasu
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200106)22:10<1946::aid-elps1946>3.0.co;2-y
Subject(s) - amplified fragment length polymorphism , dna methylation , microbiology and biotechnology , biology , dna , methylation , restriction enzyme , methylated dna immunoprecipitation , illumina methylation assay , genetics , gene , gene expression , medicine , population , genetic diversity , environmental health
We have applied a methylation‐sensitive restriction endonuclease, Not I, to the existing amplified fragment length polymorphism (AFLP) method and developed Not I‐ Mse I methylation‐sensitive‐AFLP (MS‐AFLP). Not I‐ Mse I MS‐AFLP allows the analysis of DNA methylation alterations at the Not I sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel‐eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent Not I‐ Mse I MS‐AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using Not I‐ Mse I MS‐AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.