Probe-antiprobe compositions and methods for dna or rna detection

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.

1 - 35 . (canceled) 
     
     
         36 . 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. 
     
     
         37 . The system of  claim 36 , wherein, in the probe, the first nucleotide sequence segment is disposed between the first labeling component and the second nucleotide sequence segment. 
     
     
         38 . The system of  claim 36 , wherein, in the probe, the second nucleotide sequence segment is disposed between the first labeling component and the first nucleotide sequence segment complementary to a target nucleotide sequence, thereby providing a probe-primer. 
     
     
         39 . The system of  claim 37  or  claim 38 , wherein the antiprobe sequence comprises a sequence not complementary to a plurality of different target nucleotide sequences, thereby providing a generic antiprobe suitable for a plurality of different target-specific probes. 
     
     
         40 . The system of  claim 38 , wherein the probe comprises a fluorescent donor, and wherein the second nucleotide sequence segment of the probe is not complementary to the target nucleotide sequence and comprises a cytidine-rich sequence region having about 2 to about 8 cytidines; wherein, when a target nucleotide sequence is amplified with said primer-probe, the amplicon generated comprises 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. 
     
     
         41 . The system of  claim 36 , wherein the probe is affixed to a substrate. 
     
     
         42 . 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. 
 
     
     
         43 . The method of  claim 42 , wherein the concentration of the probe-primer is about 5 nM to about 50 nM, and the concentration of the primer oligonucleotide is about 100 nM to about 600 nM.