Amplification-free detection of bacterial genes using a signaling probe-based DNA microarray

Abstract In this study, we developed a novel DNA microarray system that does not require fluorophore-labeling, amplification, or washing of the target nucleic acid fragments. Two types of DNA probes (so-called “signaling probes”) labeled with a fluorescence dye (Cy3) and quencher molecule (BHQ2) were spotted on the DNA microarray such that fluorescent signals of Cy3 could be quenched by BHQ2 due to duplex formation between the probes. The addition of the target DNA or RNA fragments disrupted the duplex formed by the probes, resulting in the generation of fluorescence signals. We examined the assay conditions of the signaling probe-based DNA microarray, including the design of the probes, hybridization temperatures, and methods for fragmentation of target molecules. Since this approach does not require time-consuming processes, including labeling, amplification, and washing, the assay achieved specific detection of 16S rDNA and 16S rRNA extracted from Escherichia coli within 60 min, which was significantly rapid compared to conventional PCR-dependent DNA microarrays.