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.

Red elemental selenium nanoparticles mediated substantial variations in growth, tissue differentiation, metabolism, gene transcription, epigenetic cytosine DNA methylation, and callogenesis in bittermelon (Momordica charantia); an in vitro experiment

“…By comparing the monitored fragment profiles of Eco RI/ Msp I and Eco RI/ Hpa II, two kinds of polymorphisms can be identified. These polymorphisms are methylation-insensitive polymorphisms displaying polymorphic patterns among samples in response to Eco RI/ Msp I and Eco RI/ Hpa II as well as methylation-sensitive polymorphisms showing the difference between Eco RI/ Hpa II and Eco RI/ Msp I profiles [ 22 ]. Comparison of MSAP fragment profiles of Eco RI/ Ms I and Eco RI/ Hpa II revealed methylation-sensitive and insensitive polymorphisms.…”

“…Genomic DNA was extracted from the leaves using a particular kit (GeneAll, South Korea). In this experiment, the Methylation-Sensitive Amplification Polymorphism (MSAP) method previously explained by Guevara et al, [ 22 ] were used to compare the profile of DNA cytosine methylation between control and a group displayed nSe toxicity. Briefly, 500 ng of the genomic DNA was subjected for digestion by Eco RI/ Hpa II or Eco RI/ Msp I conditions at 37 °C for 2h.…”

Global DNA Methylation Patterns Can Play a Role in Defining Terroir in Grapevine (Vitis vinifera cv. Shiraz)

“…Both techniques use methylation sensitive enzymes to discover DNA methylation polymorphisms between samples. Although the use of restriction enzymes has the obvious limitation of being capable of detecting such polymorphisms only on the context of their recognition sequence, the technology has been extensively validated over the last 20 years and is considered highly reliable ( Yaish et al, 2014 ; Li et al, 2015 ; Guevara et al, 2017 ).…”

Comparative epigenetic and genetic spatial structure of the perennial herb Helleborus foetidus: Isolation by environment, isolation by distance, and functional trait divergence

“…The methylation‐sensitive amplified polymorphism technique (MSAP; Schulz et al, 2013; Guevara et al, 2017) was used to characterize plants epigenetically. Genome‐wide analyses of DNA methylation in nonmodel organisms without a reference genome are still challenging.…”