Self-quenching DNA probes based on dye dimerization for identification of mycobacteria

Abstract

This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase of the fluorescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of Mycobacterium xenopi specific target at room temperature, using single-molecule detection techniques.