Abstract
A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.
Highlights
Recent advances in Next-Generation DNA sequencing [1] have led to a revolution in our understanding of the human genome
Fluorogenic probes, when used in rapid polymerase chain reaction (PCR) or real-time polymerase chain reaction (RT-PCR) assays, provide sequence specific methods to detect a range of DNA and RNA targets, to discriminate between wildtype and single-point mutations, and to analyse SNPs
During the development of an optimized oligonucleotide probe system that combines duplex stabilization with favourable fluorogenic properties we reasoned that use of intercalative and reporter dye combinations with different emission/excitation spectra would allow the probes to be used for multiplex applications, i.e. simultaneous analysis of several different targets
Summary
Recent advances in Next-Generation DNA sequencing [1] have led to a revolution in our understanding of the human genome. To take advantage of these transformative developments, the field of clinical diagnostics requires improved methods for the analysis of genomic DNA, mRNA and non-coding RNA. Such methods should be simple, sensitive and capable of high throughput. Multiple copies of the same fluorophore increase the brightness of the probe, thereby improving the limit of detection (LOD) They reduce the fluorescence in the unhybridized probe due to collisional quenching between dye molecules. This internal quenching eliminates the requirement for hairpin structures that are essential for molecular beacon function, but which complicate melting analysis and pose particular problems when attempting to target cellular RNA. Multiple dye-labelled probes such as HyBeacons are used extensively in clinical and forensic ap-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
