The Importance of Biological and Technical Controls in the Application of Methylation-Sensitive Amplified Fragment Length Polymorphism
Cunningham, Tyler Wilson
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DNA methylation is an epigenetic modification that is involved in a wide array of biological processes, including disease and aging. Methylation research involving insects has been limited in the past due to the lack of complete methylation machinery in model organisms like Drosophila melanogaster. However, a number of insects with complete sets of DNA methylation enzymes have now had their genomes sequenced, but there is still great interest in methods that assess global DNA methylation levels without genome resources. In this study, the efficacy of methylation-sensitive AFLP is investigated. Specifically, we tested this protocol s sensitivity to methylation differences and replicability. We attempted to compare methylation levels of many different organisms, such as Vespula maculifrons, Apis mellifera, Caenorhabditis elegans, Homo sapiens, and Drosophila melanogaster. In our experiments, we observed poor reproducibility between replicates. Furthermore, D. melanogaster and C. elegans, which do not exhibit significant levels of DNA methylation, exhibited particularly inconsistent and spurious results. In this study, we systematically tested each step of the methylation-sensitive AFLP protocol to identify possible sources of error. We find that, while most of the variables of the methodology are robust, some organisms and the methylation-sensitive enzyme, HpaII, do not return consistently reproducible results. Therefore, if one chooses to implement this methodology, biological and technical controls must first be proven to be working as expected before any true comparative studies may be conducted.