title: Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP).
abstract: 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.
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain.
authors: María Ángeles
authors: MÁ
authors: Guevara
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain.
authors: Nuria
authors: N
authors: de María
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain.
authors: Enrique
authors: E
authors: Sáez-Laguna
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain.
authors: María Dolores
authors: MD
authors: Vélez
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain. cervera@inia.es.
authors: María Teresa
authors: MT
authors: Cervera
authors: Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain. cabezas.joseantonio@inia.es.
authors: José Antonio
authors: JA
authors: Cabezas
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*
chemical_list: D003596:Cytosine
keywords: AFLP-based technique; Anonymous CCGG sites; Cytosine methylation; Isoschizomers; MSAP; Methylation pattern