Method of using replicatable hybridzable recombinant RNA probes
First Claim
1. A method for determining the presence or concentration of an oligo- or polynucleotide of interest in a sample, comprising the steps of:
- (a) incubating the sample with replicatable and hybridizable recombinant single-stranded RNA probe molecules comprising;
(1) a recognition sequence for the binding of an RNA-directed RNA polymerase;
(2) a sequence required for the initiation of product strand synthesis by a polymerase; and
(3) a heterologous RNA sequence (i) inserted in an externally located hairpin loop and (ii) complementary to the oligo- or polynucleotide of interest;
under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize, so as to thereby form a specific complex between a replicatable and hybridizable single-stranded RNA probe molecule and each oligo- or polynucleotide of interest in the sample;
(b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture;
(c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing complementary copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest under conditions such that the complementary copies so synthesized are separated from the recombinant RNA probe molecules, and serve as templates for synthesis of identical copies of the recombinant-RNA probe molecules;
(d) repeating step (c) at least once; and
(e) detecting complementary and identical copies of the recombinant-RNA probe molecules so synthesized in step (c), thereby determining the presence or concentration of the oligo- or polynucleotide of interest.
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Abstract
The present invention provides a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising: a recognition sequence for the binding of an RNA-directed RNA polymerase; a sequence required for the initiation of product strand synthesis by the polymerase; and a heterologus RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo or polynucleotide of interest. This invention also provides methods for determining the presence of concentration of an oligo- or polynucleotide of interest in a sample and for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample.
47 Citations
26 Claims
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1. A method for determining the presence or concentration of an oligo- or polynucleotide of interest in a sample, comprising the steps of:
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(a) incubating the sample with replicatable and hybridizable recombinant single-stranded RNA probe molecules comprising; (1) a recognition sequence for the binding of an RNA-directed RNA polymerase; (2) a sequence required for the initiation of product strand synthesis by a polymerase; and (3) a heterologous RNA sequence (i) inserted in an externally located hairpin loop and (ii) complementary to the oligo- or polynucleotide of interest; under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize, so as to thereby form a specific complex between a replicatable and hybridizable single-stranded RNA probe molecule and each oligo- or polynucleotide of interest in the sample; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing complementary copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest under conditions such that the complementary copies so synthesized are separated from the recombinant RNA probe molecules, and serve as templates for synthesis of identical copies of the recombinant-RNA probe molecules; (d) repeating step (c) at least once; and (e) detecting complementary and identical copies of the recombinant-RNA probe molecules so synthesized in step (c), thereby determining the presence or concentration of the oligo- or polynucleotide of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample, comprising the steps of:
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(a) incubating the sample with a mixture of different types of replicatable and hybridizable recombinant single-stranded RNA probe molecules comprising; (1) a recognition sequence for the binding of an RNA-directed RNA polymerase; (2) a sequence required for the initiation of product strand synthesis by a polymerase; and (3) a heterologous RNA sequence (i) inserted in an externally located hairpin loop and (ii) complementary to one of the oligo- or polynucleotides of interest each type having a different inserted sequence, under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize, so as to thereby form specific complexes between replicatable and hybridizable recombinant single-stranded RNA probe molecules from the mixture and the oligo- or polynucleotides of interest in the sample; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing complementary copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotides of interest under conditions such that the complementary copies so synthesized are separated from the recombinant-RNA probe molecules, and serve as templates for synthesis of identical copies of the recombinant-RNA probe molecules; (d) repeating step (c) at least once; (e) separating the mixture of the complementary and identical copies of the recombinant-RNA probe molecules so synthesized by hybridizing them to an ordered array of polynucleotides bound to a solid support, where each of the polynucleotides is complementary to one type of the complementary or identical copies of the recombinant-RNA probe molecules so synthesized; and (f) detecting the complementary or identical copies of the recombinant-RNA probe molecules separated in step (e), thereby determining the presence or concentration of each oligo- or polynucleotide of interest. - View Dependent Claims (23, 24, 25, 26)
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Specification