Probe-antiprobe compositions and methods for dna or rna detection
AssigneeGenetag Technology, Inc.
InventorDavid A. Shafer
Life of this patent
Estimating the term…
Abstract
The invention provides novel compositions and methods for detecting unlabeled nucleic acid targets using labeled polynucleotide probes and partially complementary antiprobes. The interaction of probes, antiprobes and targets result in signaling changes that indicate target frequency. This novel detection mechanism is called a DNA detection switch, and it enable end-point detection, microarray detection and real-time PCR detection of a variety of nucleic acid targets including microbial species and subspecies, drug resistant mutants, and pathogenic strains.
Claims9
1-35. (canceled)
A probe-antiprobe system comprising:
- (a) a probe comprising a first labeling component, a first nucleotide sequence segment complementary to a target nucleotide sequence, and a second nucleotide sequence segment not complementary to a target nucleotide sequence;
- and
- (b) an antiprobe comprising a second labeling component and a nucleotide sequence complementary to the second nucleotide sequence of the probe;
- wherein the first and second labeling components are a fluorescent donor-acceptor pair, wherein the fluorescent donor is a fluorophore, and the fluorescent acceptor is selected from the group consisting of:
- a quencher, a second fluorophore, and a guanine-rich nucleotide sequence comprising about 2 to about 8 guanines, and wherein the system exhibits a first signaling state when the first and second labeling components are in proximity due to binding of the probe to the antiprobe in the absence of a target nucleotide sequence, and exhibits a second signaling state when the first and second labeling components are dissociated when the probe binds to a target nucleotide sequence.
A method of quantitative real-time PCR, the method comprising the steps of:
- (a) obtaining a sample suspected of having a target nucleotide sequence;
- (b) obtaining a PCR amplification reaction mix comprising:
- (i) the sample;
- (ii) a primer-probe comprising:
- (1) a 5′ fluorescent donor, (2) a second nucleotide sequence segment not complementary to the target sequence that further comprises a cytidine-rich region having about 2 to about 8 cytidines, and (3) a 3′ primer sequence segment that is specific to a first region of the target nucleotide sequence,
- (iii) and a primer oligonucleotide that is specific to a second region of the target nucleotide sequence,
- wherein the ratio of the concentration of the primer-probe to the concentration of the primer oligonucleotide is between about 1:2 to about 1:125;
- (c) calculating the number of target copies that can be made with the restricted quantity of primer-probes;
- (d) amplifying the target nucleotide sequence encompassed by the primer-probe and the primer, thereby generating an amplicon comprising an antiprobe, wherein said antiprobe sequence is complementary to the second nucleotide sequence of the primer-probe, and wherein the antiprobe further comprises a fluorescent acceptor comprising a guanine-rich sequence region comprising about 2 to about 8 guanines;
- and
- (e) monitoring the signaling profile to determine a cycling time point indicating exhaustion of the primer-probes provided, thereby providing a quantitative indicator of the number of amplified targets generated relative to the number of amplification cycles completed.
Dependencies are inferred from the claim text.