Diagnostic applications of double D-loop formation
First Claim
1. A diagnostic method for detecting a linear duplex DNA analyte, having first and second strands, containing a first internal DNA target sequence, comprisingproviding 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 ATPγ
- S, rATP, dATP, and GTPγ
S, or a mixture of nucleotide cofactors consisting of ATPγ
S and rATP or ATPγ
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, anddetecting the presence of the probe DNA in the probe;
target complex.
5 Assignments
0 Petitions
Accused Products
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.
111 Citations
21 Claims
-
1. 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 ATPγ - S, rATP, dATP, and GTPγ
S, or a mixture of nucleotide cofactors consisting of ATPγ
S and rATP or ATPγ
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, anddetecting the presence of the probe DNA in the probe;
target complex. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- S, rATP, dATP, and GTPγ
-
8. 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, andisolating the duplex DNA analyte containing the target sequence from the probe;
target complex. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
- target complex containing the probe strands and both target strands, where said complex is stable to deproteinization,
Specification