Detection of nucleic acids by target-catalyzed product formation

Abstract

A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5′-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5′-nuclease to cleave the oligonucleotide to provide (i) a first fragment-that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3′ of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte. The method has particular application to the detection of a polynucleotide analyte such as DNA. Kits for conducting methods in accordance with the present invention are also disclosed.

1 - 34 . (canceled)  
     
     
         35 . A kit for detection of a polynucleotide comprising in packaged combination: 
 (a) a first oligonucleotide having the characteristic that, when reversibly hybridized under isothermal conditions to at least a portion of the polynucleotide, it is degraded by a 5′-nuclease to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide and (ii) a second fragment that is 3′ of the first fragment in the first oligonucleotide and is substantially hybridizable to the polynucleotide, wherein the isothermal conditions are at or near the melting temperature of a duplex comprising the second fragment hybridized to the polynucleotide, and    (b) a second oligonucleotide having the characteristic of hybridizing to a site on the polynucleotide that is separated by no more than five nucleotides from the 3′-end of the site at which the second fragment of the first oligonucleotide hybridizes, wherein the polynucleotide is substantially fully hybridized to the second oligonucleotide at said melting temperature.    
     
     
         36 . The kit of  claim 35  wherein the second oligonucleotide hybridizes to a site on the polynucleotide that is separated by no more than one nucleotide from the 3′-end of the site at which the second fragment of the first oligonucleotide hybridizes.  
     
     
         37 . The kit of  claim 35 , wherein the second oligonucleotide hybridizes to a site on the polynucleotide that is contiguous with the 3′-end of the site at which the second fragment of the first oligonucleotide hybridizes.  
     
     
         38 . The kit of  claim 35 , wherein the second oligonucleotide dissociates from the duplex at a temperature at least 3° C. higher than the temperature of said melting point.  
     
     
         39 . The kit of  claim 35 , wherein the second oligonucleotide dissociates from the duplex at a temperature at least 5° C. higher than the temperature of said melting point.  
     
     
         40 . The kit of  claim 35 , wherein the second oligonucleotide is at least as long as the second fragment of the first oligonucleotide.  
     
     
         41 . The kit of  claim 35 , wherein the second oligonucleotide is at least 2 nucleotides longer than the second fragment of the first oligonucleotide.  
     
     
         42 . The kit of  claim 35 , further comprising a label.  
     
     
         43 . The kit  claim 35 , wherein the first fragment of the first oligonucleotide comprises a label.  
     
     
         44 . The kit of  claim 35 , further comprising a buffering media.  
     
     
         45 . The kit of  claim 35 , further comprising a 5′-nuclease.  
     
     
         46 . The kit of  claim 45 , wherein the 5′-nuclease comprises a 5′-endonuclease.  
     
     
         47 . The kit of  claim 45 , wherein the 5′-nuclease comprises a Taq polymerase.