Methods for detection of a target nucleic acid by capture using multi-subunit probes

Abstract

The invention relates to generating a signal indicative of a target nucleic acid sequence, comprising forming a complex by incubating a sample comprising a target nucleic acid sequence with a probe comprising a first and second subunit, and a binding moiety, and dissociating the first and second subunit to release the first subunit and generate a signal. The invention also relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a complex by incubating a target nucleic acid sequence, an upstream primer and a probe comprising a first and second subunit, and a binding moiety. The primer is extended with a nucleic acid polymerase to displace a portion of the first subunit from the target nucleic acid strand thereby dissociating the first subunit from the second subunit to release the first subunit and generate a signal.

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         2 . A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising 
 forming a detection complex by incubating a sample comprising a target nucleic acid sequence, and a probe, wherein said probe comprises at least a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety,    binding said probe to said target nucleic acid sequence wherein said first subunit of said probe dissociates from at least said second subunit of said probe to generate a signal,    wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and    wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.    
     
     
         3 . The method of  claim 2 , wherein the sample further comprises an upstream primer wherein said upstream primer hybridizes to said target nucleic acid sequence upstream of where said probe hybridizes to said target nucleic acid sequence and wherein the method further comprises 
 subjecting said detection complex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand, and displacement of said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit dissociates from at least said second subunit of said probe and is released to generate a signal,    wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand at or below said polymerization temperature.    
     
     
         4 . The method of  claim 2  wherein said binding moiety is a tag.  
     
     
         5 . The method of  claim 2  wherein said binding moiety is a nucleic acid sequence that binds to a capture element.  
     
     
         6 . The method of  claim 2  wherein said signal is detected or measured, and wherein said detecting and/or measuring the signal comprises detecting and/or measuring the amount of said released first subunit captured by binding of said binding moiety to a capture element on a solid support.  
     
     
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         11 . The method of  2 , wherein a detection complex is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when the probe is not bound to said target nucleic acid or when said first subunit of the probe is not dissociated from said second subunit of the probe.  
     
     
         12 . The method of  claim 11  wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.  
     
     
         13 . The method of  claim 2 , wherein said first subunit of said probe further comprises a reporter.  
     
     
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         15 . The method of  claim 4 , wherein said released first subunit of the probe is captured by binding of said tag to a capture element.  
     
     
         16 . The method of  claim 2 , wherein said first subunit is 5 to 10,000 nucleotides in length.  
     
     
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         24 . The method of  claim 2 , wherein said probe comprises a target nucleic acid binding sequence comprising the combination of a region of two or more subunits of said probe.  
     
     
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         27 . The method of  claim 2 , wherein said subunits are covalently bound to each other when not bound to said target nucleic acid sequence.  
     
     
         28 . The method of  claim 2 , wherein said subunits are non-covalently bound to each other when not bound so said target nucleic acid sequence.  
     
     
         29 . A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising: 
 (a) providing a detection complex comprising a probe, wherein said probe comprises at least a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of said target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and    b) amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within said target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and thereby dissociates said first subunit of said probe from at least said second subunit of said probe thereby creating detectable, released labeled first subunits of said probe;    wherein said amplification is performed at an amplification temperature and said first subunit of said probe does not dissociate from at least said second subunit of said probe when not bound to said target nucleic acid sequence at or below said amplification temperature; and    c) detecting and/or measuring the amount of released, labeled first subunit of said probe captured by binding of said binding moiety to a capture element on a solid support as an indicator of the presence of the target sequence in the sample.    
     
     
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         48 . A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a probe, wherein said probe comprises at least a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, and packaging means thereof, 
 wherein said probe can bind to a target nucleic acid sequence to form a detection complex; and    wherein said binding is performed at a binding temperature, and wherein said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.    
     
     
         49 . The kit of  claim 48 , further comprising an upstream primer and a nucleic acid polymerization activity, wherein said upstream primer hybridizes to said target nucleic acid sequence upstream of where said probe hybridizes to said target nucleic acid sequence and 
 wherein said upstream primer and said probe can bind to a target nucleic acid sequence to form a detection complexed.    
     
     
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         51 . The kit of  claim 48 , wherein said first subunit of said probe comprises a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal when said probe is not bound to said target nucleic acid or when said first subunit of said probe is not dissociated from said second subunit of the probe.  
     
     
         52 . The kit of  claim 51 , wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.  
     
     
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         56 . The kit of  claim 48  wherein said subunit is 8 to 500 nucleotides in length.  
     
     
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         65 . The kit of  claim 48  wherein said first and second subunits are non-covalently bound to each other when not bound to said target nucleic acid sequence.