Methylation interference

Copyright information:Taken from "Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme"Nucleic Acids Research 2006;33(22):7164-7175.Published online 3 Jan 2006PMCID:PMC1325019.© The Author 2005. Published by Oxford University Press. All rights reserved () and () outline the experimental scheme for obtaining DNA ladders to be analyzed as ‘Lane C’ and ‘Lane T’ in (). Briefly, the 3′-cleavage fragment obtained from EC2 treated with DMS before (Test-T) or after (Control-C) the cleavage reaction was performed and labeled at the 5′ end with P. Under our reaction condition, DMS only methylated the N7 atom of one guanine per deoxyribozyme molecule on average. Methylated guanines were cleaved by piperidine at 90°C and cleaved fragments were resolved by 10% denaturing PAGE. Methylated guanines that disrupt deoxyribozyme activity appear missing or lighter in lane T than lane C. Filled black circles at the left side of the gel (and next to a concerned nucleotide) indicate the DNA bands with significantly reduced intensity in T lanes while the black square labels the DNA band that had a significantly enhanced intensity in the T lane. EC2 was used for this experiment. The sequence of the cleavage fragment P2 in (C) was drawn using the same labeling scheme given in .