JUNCTION PROBES - SEQUENCE SPECIFIC DETECTION OF NUCLEIC ACIDS VIA TEMPLATE ENHANCED HYBRIDIZATION PROCESSES

Abstract

As new disease biomarkers such as cancer-linked microRNAs are discovered, the need for new strategies for the detection of these disease biomarkers at isothermal conditions will increase. The junction probe (JP) technology is a restriction endonuclease-based nucleic acids detection platform that achieves isothermal amplified sensing and does not require the presence of an endonuclease recognition sequence in the target analyte. The first generation junction probe platform however suffered from long assay time (several hours). We hypothesized that the slow catalysis in the first-generation JP platform was due to an inhibition cycle. Consequently, a second generation JP platform, which is modified with phosphorothioate moieties in order to suppress the inhibition cycle, was developed. The second-generation JP platform is significantly superior to the first-generation platform and we demonstrated the potential of second-generation JP by detecting microRNA and bacterial ribosomal RNA. Importantly, second-generation JP could detect RNA in crude bacterial cell lysates without extensive sample preparation.