Diagnostic applications of double D-loop formation
AssigneeDaikin Industries, Ltd.
InventorsElissa P. Sena, Cornelia J. Calhoun, David A. Zarling
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Abstract
The present invention describes the formation of RecA protein catalyzed double-stranded probe:duplex linear target DNA complexes that are stable to deproteinization. The uses of this stable probe:target complex in diagnostic/DNA detection systems in in vitro and in situ DNA hybridization reactions is discussed. The probe:target complexes are also useful for diagnostic application in RecA protein facilitated DNA amplification reactions.
Claims21
A diagnostic method for detecting a linear duplex DNA analyte, having first and second strands, containing a first internal DNA target sequence, comprising
- providing a set of two DNA probes, having first and second probe strands, where the first and second probe strands (i) contain complementary sequences to the first and second target sequence strands, and (ii) where these complementary sequences also contain complementary overlap between the probe strands,
- coating the probes with RecA protein in a RecA protein coating reaction, said coating reaction containing a nucleotide cofactor selected from the group consisting of ATP.gamma.S, rATP, dATP, and GTP.gamma.S, or a mixture of nucleotide cofactors consisting of ATP.gamma.S and rATP or ATP.gamma.S and ADP,
- combining the RecA coated probes with the linear duplex DNA, which contains the target sequence, under conditions that produce a probe:target complex containing the probe strands and both target strands, where said complex is stable to deproteinization, and
- detecting the presence of the probe DNA in the probe:target complex.
A method for isolating a linear duplex DNA analyte, having first and second strands, containing a first internal DNA target sequence, where said duplex DNA analyte is present in a mixture of nucleic acid molecules, comprising
- providing a set of two DNA probes, having first and second probe strands, where the first and second probe strands (i) contain complementary sequences to the first and second target sequence strands, and (ii) where these complementary sequences also contain complementary overlap between the probe strands,
- coating the probes with RecA protein in a RecA protein coating reaction,
- combining the RecA coated probes with the linear duplex DNA, which contains the target sequence, under conditions that produce a probe:target complex containing the probe strands and both target strands, where said complex is stable to deproteinization,
- separating the probe:target complex from the mixture of nucleic acid molecules, and
- isolating the duplex DNA analyte containing the target sequence from the probe:target complex.
Dependencies are inferred from the claim text.