Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification

N6-methyladenine (6mA) is reported as a potential epigenetic marker in eukaryotic genomes. However, accurate identification of the location of 6mA in DNA remains a challenging task. Here, we show that Ag⁺ can selectively stabilize the A-C mismatch and efficiently promote primer extension. In contrast, the complex of 6mA-Ag⁺-C is instable and therefore cannot be recognized by DNA polymerases, resulting in the termination of primer extension. Based on this finding, we successfully identified and quantified 6mA at the single-base level through the analysis of gel bands of extended primers and fluorescence measurements combined with rolling circle amplification. The high selectivity and sensitivity of this strategy may provide a new platform for the efficient analysis of 6mA in DNA in the future.