Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.
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.
Ruiz-García
Leonor
L
Departamento de Biotecnología y Protección de Cultivos, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Murcia, Spain.
Cabezas
Jose Antonio
JA
de María
Nuria
N
Cervera
María-Teresa
MT
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D018744:DNA, Plant / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry; D019003:Deoxyribonuclease HpaII / Q000737:chemistry; D010944:Plants; D012150:Polymorphism, Restriction Fragment Length; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry
D018744:DNA, Plant; D003596:Cytosine; C020098:DNA modification methylase EcoRI; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D019003:Deoxyribonuclease HpaII