Visual DNA amplification using a simple PCR device is useful for field tests to detect target DNA and RNA. We hereby de-scribe a detection system involving PCR amplification visual-ized with the naked eye, by genetic alphabet expansion. The system employs fluorescence resonance energy transfer (FRET) between unnatural base combinations: self-quenched dinucleo-tides of 2-amino-6-(2-thienyl)purine (s) as a donor and Cy3-conjugated 2-nitro-4-propynylpyrrole (Cy3-hx-Px) as an accep-tor. During PCR, the triphosphate substrate of Cy3-hx-Px (Cy3-hx-dPxTP) is incorporated into DNA opposite its pairing part-ner, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), in the primer, which also contains the dinucleotides of s. Thus, the amplified DNA can be visualized by the Cy3 fluorescence resulting from the FRET between the s-dinucleotides and the incorporated Cy3-hx-Px upon 365 nm irradiation. Using this system, we demonstrated the visual SNP detection of a series of quinolone-resistant bacteria genes.
This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) (to M.K. and I.H.).