Silver nanoclusters (AgNCs) stabilized by double-stranded DNA (dsDNA) are of special interest because the duplex structures provide rigidity and chirality that can be transferred to the metal clusters. This work reports fluorescent AgNCs obtained from dsDNA templates containing artificial ligand-derived nucleobases. They are compared with fluorescent AgNCs obtained from DNA templates containing C:C mismatches (C, cytosine). Towards this end, the new metal-mediated Im-Ag(I) -C base pair composed of imidazole (Im) and cytosine was introduced into dsDNA and reduced to form AgNCs. The clusters were characterized by UV/Vis, fluorescence and circular dichroism spectroscopy. The AgNCs form chiral aggregates with dsDNA. Their optical properties are highly sequence-dependent. As a result, a simple method to detect a cytosine insertion into a run of cytosine residues in a duplex is proposed using the human CDH1 gene as an example.