Hydrolysis probes

Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In one embodiment, a sample is contacted with a primer complementary to a first region of a target nucleic acid and a probe complementary to a second region of the target nucleic acid downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the primer and the probe. The probe in this embodiment comprises a fluorophore and is attached to a solid support. The hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support. The presence of the target nucleic acid is then detected and optionally quantified by detecting a decrease in signal from the reporter on the solid support.

1 - 75 . (canceled) 
     
     
         76 . A method for quantifying an amount of a target nucleic acid in a sample, comprising:
 (a) amplifying the target nucleic acid in the presence of a nucleic acid polymerase having exonuclease activity, a target-specific primer pair comprising a first primer complementary to a first region on a first strand of the target nucleic acid and a second primer complementary to a region on a second strand of the target nucleic acid, and a target-specific probe complementary to a second region on the first strand of the target nucleic acid downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the target-specific primer and the target-specific probe, wherein the target-specific probe comprises a tag at its 5′ or 3′ end and a reporter, and further wherein the nucleic acid polymerase cleaves the target-specific probe and releases the reporter from a solid support when extending the first primer along the first strand of the target nucleic acid;   (b) detecting a first signal from the reporter on the solid support at a first time and a second signal from the reporter on the solid support at a second time; and   (c) correlating a change in signal with the amount of the target nucleic acid in the sample.   
     
     
         77 . The method of  claim 76 , wherein quantifying the amount of the target nucleic acid in the sample comprises using a standard curve. 
     
     
         78 . The method of  claim 76 , wherein quantifying the amount of the target nucleic acid in the sample comprises determining a relative amount of the target nucleic acid. 
     
     
         79 . The method of  claim 76 , further comprising detecting at least a third signal from the reporter on the solid support at a third time. 
     
     
         80 . The method of  claim 76 , comprising detecting a signal from the reporter on the solid support prior to extending the first primer with the nucleic acid polymerase having exonuclease activity to cleave the hybridized target-specific probe and release the reporter from the solid support. 
     
     
         81 . The method of  claim 76 , wherein the solid support is an encoded bead. 
     
     
         82 . The method of  claim 76 , comprising quantifying an amount of a plurality of different target nucleic acids in the sample 
     
     
         83 . The method of  claim 76 , wherein the reporter is a fluorphore. 
     
     
         84 . The method of  claim 83 , wherein the fluorphore is attached at the 5′ end of the target-specific probe. 
     
     
         85 . The method of  claim 83 , wherein the change in the signal is a decrease in a fluorescent signal. 
     
     
         86 . The method of  claim 76 , wherein the reporter is a fluorphore and quencher pair. 
     
     
         87 . The method of  claim 86 , wherein the change in the signal is an increase in a fluorescent signal. 
     
     
         88 - 118 . (canceled)