Abstract
BackgroundDNA methylation plays a key role in development, contributes to genome stability, and may also respond to external factors supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA.ResultsHere, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII + MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile.ConclusionsWe recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation.
Highlights
DNA methylation plays a key role in development, contributes to genome stability, and may respond to external factors supporting adaptation and evolution
Principle of Methylation Sensitive Amplified Polymorphism (MSAP) In the original MSAP protocol [22], genomic DNA is divided into two parts, and each part is digested with EcoRI, which recognizes the GAATTC target site and is thought to be negligibly influenced by DNA cytosine methylation, and one of the methylation-sensitive MspI or HpaII isoschizomers, which can both cleave CCGG sequences
The DNA samples digested with EcoRI and MspI or EcoRI and HpaII are ligated to two dsDNA adapters compatible with EcoRI- and MspI/HpaII-generated ends
Summary
DNA methylation plays a key role in development, contributes to genome stability, and may respond to external factors supporting adaptation and evolution. Electrophoretic patterns produced by the method are rather difficult to interpret, when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. The DNA of most eukaryotic organisms contains 5methylcytosine (mC) residues, which represent important epigenetic information involved in the regulation of gene expression during various developmental processes such as cell differentiation, imprinting, or X chromosome inactivation [1,2,3]. DNA methylation contributes to genome stability by repressing harmful genetic elements [4]. The level of mC varies among individual species, and mC distribution along DNA is not uniform
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