Ruiz-García Leonor, Cabezas Jose Antonio, de María Nuria, Cervera María-Teresa
Departamento de Biotecnología y Protección de Cultivos, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Murcia, Spain.
Methods Mol Biol. 2010;631:63-74. doi: 10.1007/978-1-60761-646-7_7.
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.
已经开发出不同的分子技术来研究甲基化胞嘧啶的整体水平或特定基因序列的甲基化情况。其中之一是对扩增片段长度多态性（AFLP）技术的一种改进，该技术已被用于研究不同真菌、植物和动物物种中匿名CCGG序列的甲基化。这项技术的主要变化基于使用具有不同甲基化敏感性的同裂酶（如HpaII和MspI）作为常用的切割限制性酶。对于每个样本，使用EcoRI/HpaII和EcoRI/MspI消化的样本进行AFLP分析。EcoRI/HpaII和EcoRI/MspI片段模式之间的比较分析允许识别两种类型的多态性：（1）“甲基化不敏感多态性”，其显示常见的EcoRI/HpaII和EcoRI/MspI模式，但在样本中被检测为多态性扩增片段；（2）“甲基化敏感多态性”，其与EcoRI/HpaII和EcoRI/MspI模式之间存在或不存在或强度不同的扩增片段相关。本章描述了该技术的详细方案，并讨论了可用于将该技术调整到不同目标物种的修改方法。
