Protocols AFLP-Based Detection of DNA Methylation

Abstract. By using the isoschizomers Hpa II and Msp I which display differential sensitiv-ity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organ-isms. Genomic DNA was digested with a mixture of EcoR 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 be-tween Hpa II and Msp 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 and in 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 or in vitro shoots were observed. These results demonstrated that this protocol is ef-fective in identifying methylated DNA profiles. Key words: amplified fragment length polymorphism (AFLP), apple, isoschizomer, methylatio