AFLP-Based detection of DNA methylation
- 760 Downloads
By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles.
Key wordsamplified fragment length polymorphism (AFLP) apple isoschizomer methylation
Unable to display preview. Download preview PDF.
- Rossi V, Motto M and Pelligrini L (1997) Analysis of the methylation pattern of the maizeOpaque-2 (O2) promoter and in vitro binding studies indicate that the O2 B-Zip protein and other endosperm factors can bind to methylated target sequences. J Biol Chem 272: 13758–13765.PubMedCrossRefGoogle Scholar
- Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T and Nagao M (1997) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA 94: 2284–2289.PubMedCrossRefGoogle Scholar