title: How to interpret methylation sensitive amplified polymorphism (MSAP) profiles?
abstract: BACKGROUND
  DNA 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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v,v,i,, Královopolská 135, Brno CZ-612 65, Czech Republic. fulnecek@ibp.cz.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Fulneček
authors.forename: Aleš
authors.initials: A
authors.lastname: Kovařík
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D000818:Animals; D019175:DNA Methylation; D018744:DNA, Plant / Q000235:genetics; D044127:Epigenesis, Genetic; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011110:Polymorphism, Genetic; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics; D014714:Vertebrates / Q000235:genetics
chemical_list: D018744:DNA, Plant; D044503:5-Methylcytosine