A homogeneous nucleic acid hybridization assay based on stand displacement

Abstract

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement.

RNA-DNA hybridization promoted by E.coli RecA portein

“…The gels were then dried and subjected to autoradiography. Native unhybridized RNA and RNA in hybrid form were readily distinguishable, as the migration of hybridized RNA was retarded (24). Quantitation was done by direct scanning of the dried gel with a Betascope 603 blot analyzer (Betagen, Waltham, MA).…”

Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments

“…Early experimental efforts used chromatography to monitor the displacement of a shorter DNA strand (1.6 Kb) by a much longer strand (6.1 Kb) in oligonucleotide solutions ( 24 ). In other early studies, the duplex region of the probe was comprised of an RNA–DNA hybrid in which the displaced RNA was detected through a multistep bioluminescence approach ( 25 , 26 ). Related experimental efforts incorporated intentional single base-pair mismatches within the duplex region of the dsProbes ( 27 ).…”

“…While long, single-stranded toehold regions were characteristic of many early studies, as few as three bases are needed to establish a stable duplex nuclei and facilitate the branch migration that precedes displacement of the original hybridization partner ( 46 ). While strand displacement-based approaches continue to gain popularity in the rational design of many systems, the discussion of strand displacement kinetics is less prevalent and focuses almost exclusively on oligonucleotide solutions in which both the dsProbes and targets are soluble species ( 24 , 25 , 27 , 29 , 47 ). Recent reports by Zhang and Winfree ( 48 ) and Genot et al .…”

Rapid SLT Gene Detection on Polyethylene–Coacrylic Acid Film without Molecular Labels or Surface-Fouling Agents

“…Because the gel separation indicated a successful amplification of the SLT-II gene, we postulated that the negative result was caused either by competition from homogeneous hybridization of the target or by slow displacement of captured target from the filmattached oligo probe. A displacement reaction could be driven by formation of the longer, and hence more stable, homogeneous product (14). Competition from homogeneous hybridization seems likely when one considers that the surface concentration of probe must be quite low, since the surface density of carboxylate groups on PEAA is low, and the target concentration of SLT-II would be high after PCR.…”