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Plant Molecular Biology Reporter

, Volume 18, Issue 4, pp 361–368 | Cite as

AFLP-Based detection of DNA methylation

  • Mingliang Xu
  • Xiangqian Li
  • Schuyler S. Korban
Protocols

Abstract

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 words

amplified fragment length polymorphism (AFLP) apple isoschizomer methylation 

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Copyright information

© Springer 2000

Authors and Affiliations

  • Mingliang Xu
    • 1
  • Xiangqian Li
    • 1
  • Schuyler S. Korban
    • 1
  1. 1.Department of Natural Resources and Environmental SciencesUniversity of IllinoisUrbana

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