Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP)

  • María Ángeles Guevara
  • Nuria de María
  • Enrique Sáez-Laguna
  • María Dolores Vélez
  • María Teresa CerveraEmail author
  • José Antonio CabezasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1456)


Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Key words

MSAP AFLP-based technique Isoschizomers Cytosine methylation Anonymous CCGG sites Methylation pattern 


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • María Ángeles Guevara
    • 1
  • Nuria de María
    • 1
  • Enrique Sáez-Laguna
    • 1
  • María Dolores Vélez
    • 1
  • María Teresa Cervera
    • 1
    Email author
  • José Antonio Cabezas
    • 1
    Email author
  1. 1.Department of Forest Ecology and GeneticInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR)MadridSpain

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