Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP)

Mahmoud W. Yaish, Mingsheng Peng, Steven J. Rothstein

Research output: Chapter in Book/Report/Conference proceedingChapter

28 Citations (Scopus)

Abstract

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages285-298
Number of pages14
Volume1062
ISBN (Print)9781627035798
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1062
ISSN (Print)10643745

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Keywords

  • 5-azaC and zebularine
  • DNA methylation
  • MSAP
  • Mutant lines

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Yaish, M. W., Peng, M., & Rothstein, S. J. (2014). Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). In Methods in Molecular Biology (Vol. 1062, pp. 285-298). (Methods in Molecular Biology; Vol. 1062). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-580-4_16