Development of fluorescence-based nucleic acid blot hybridization method using Cy5.5 labeled DNA probes

Abstract

Near-infrared (NIR) fluorophores are widely used as fluorescent probes for bioimaging because of their minimal photodamage to biological samples, deep penetration, and low interference from background autofluorescence. Here, we employed a NIR fluorescent cyanine dye Cy5.5 to label DNA probes for nucleic acid blot hybridization. The specificity and sensitivity of fluorescent DNA probes were proven by both Southern blot and Northern blot using cellulolytic bacterium Ruminiclostridium cellulolyticum as a model. Furthermore, employing the method, we successfully identified the gene disruption of ClosTron to rule out off-target, analyzed the differential transcription of genes under different conditions, and confirmed RNA cleavage. Compared to other nonradioactive probes, the preparation and detection of Cy5.5-labeled probes are more simple, more economical, and versatile, suggesting that the Cy5.5-labeled probes are suitable for nucleic acid blot hybridization in addition to bioimaging.