Isoschizomers and Amplified Fragment Length Polymorphism for the Detection of Specific Cytosine Methylation Changes

  • Leonor Ruiz-García
  • Jose Antonio Cabezas
  • Nuria de María
  • María-Teresa CerveraEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)


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 a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. 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” that are associated with amplified fragments differing 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 modifications that can be applied to adjust the technology to different species of interest.

Key words

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


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Leonor Ruiz-García
    • 1
  • Jose Antonio Cabezas
    • 2
  • Nuria de María
    • 3
  • María-Teresa Cervera
    • 3
    Email author
  1. 1.Departamento de Biotecnología y Protección de CultivosInstituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA)MurciaSpain
  2. 2.Departamento de Investigación AgroalimentariaInstituto Madrileño de Investigación y Desarrollo Rural, Agrario y AlimentarioAlcalá de HenaresSpain
  3. 3.Departamento de Sistemas y Recursos ForestalesCIFORMadridSpain

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