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 . A method for detecting a target nucleic acid in a sample, comprising:
 (a) contacting the sample with a first target-specific primer complementary to a first region on a first 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 first target-specific primer and the target-specific probe, wherein the target-specific probe comprises a reporter and is attached to a solid support;   (b) cleaving the hybridized target-specific probe with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support; and   (c) detecting the target nucleic acid by detecting a change in signal from the reporter on the solid support.   
     
     
         2 . The method of  claim 1 , wherein the first target-specific primer and the target-specific probe hybridize to adjacent sequences on the target nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein the first target-specific primer and the target-specific probe hybridize to non-adjacent sequences on the target nucleic acid. 
     
     
         4 . The method of  claim 1 , further comprising extending the first target-specific primer with the nucleic acid polymerase having exonuclease activity. 
     
     
         5 . The method of  claim 1 , wherein the solid support is an encoded bead. 
     
     
         6 . The method of  claim 1 , wherein the reporter is a fluorphore. 
     
     
         7 . The method of  claim 6 , wherein the fluorphore is attached at the 5′ end of the target-specific probe. 
     
     
         8 . The method of  claim 6 , wherein the change in the signal is a decrease in a fluorescent signal. 
     
     
         9 . The method of  claim 1 , wherein the reporter is a fluorphore and quencher pair. 
     
     
         10 . The method of  claim 9 , wherein the change in the signal is an increase in a fluorescent signal. 
     
     
         11 . The method of  claim 1 , further comprising detecting a reference signal from a reporter on a non-hybridizing probe attached to a solid support. 
     
     
         12 . The method of  claim 11 , wherein the non-hybridizing probe is attached to a spatially discrete location on the same solid support to which the target-specific probe is attached. 
     
     
         13 . The method of  claim 11 , wherein the non-hybridizing probe is attached to a different solid support than that to which the target-specific probe is attached. 
     
     
         14 . The method of  claim 13 , wherein the different solid supports are different encoded beads. 
     
     
         15 . The method of  claim 1 , wherein the target nucleic acid is a first target nucleic acid, the reporter is a first reporter, the solid support is a first solid support, and the method further comprises:
 (a) contacting the sample with a second target-specific primer complementary to a first region on a first strand of a second target nucleic acid, and a second target-specific probe complementary to a second region on the first strand of the second target nucleic acid downstream of the first region under conditions suitable for hybridization of the second target nucleic acid with the second target-specific primer and the second target-specific probe, wherein the second target-specific probe comprises a second reporter and is attached to a second solid support;   (b) cleaving the second hybridized target-specific probe with the nucleic acid polymerase having exonuclease activity to release the second reporter from the second solid support; and   (c) detecting the second target nucleic acid by detecting a change in signal from the second reporter on the second solid support.   
     
     
         16 . The method of  claim 15 , wherein the first solid support and the second solid support are spatially discrete locations on one solid support. 
     
     
         17 . The method of  claim 15 , wherein the first solid support is physically separate from the second solid support. 
     
     
         18 . The method of  claim 15 , wherein the first reporter and the second report are the same. 
     
     
         19 . The method of  claim 15 , wherein the first reporter and the second reporter are different. 
     
     
         20 . The method of  claim 11 , further comprising normalizing for changes in fluorescence over time. 
     
     
         21 . The method of  claim 1 , further comprising contacting the sample with a second target-specific primer complementary to a region on a second strand of the target nucleic acid. 
     
     
         22 . The method of  claim 21 , further comprising performing multiple polymerase chain reaction cycles. 
     
     
         23 . The method of  claim 22 , wherein the multiple polymerase chain reaction cycles are performed without a wash step to remove free-floating fluorophore between cycles. 
     
     
         24 . The method of  claim 22 , wherein detecting the change in signal from the reporter on the solid support comprises detecting the signal before and after performing the multiple polymerase chain reaction cycles. 
     
     
         25 . The method of  claim 22 , wherein detecting the change in signal from the reporter on the solid support comprises detecting the signal only after performing the multiple polymerase chain reaction cycles. 
     
     
         26 . The method of  claim 25 , further comprising comparing the detected signal from the reporter on the solid support to a predetermined ratio of the signal of the reporter on the solid support to a reference signal from a reporter on a non-hybridizing probe attached to a solid support. 
     
     
         27 . The method of  claim 22 , further comprising quantifying the amount of the target nucleic acid in the sample. 
     
     
         28 . The method of  claim 27 , wherein quantifying the amount of the nucleic acid target in the sample comprises using a standard curve. 
     
     
         29 . The method of  claim 27 , wherein quantifying the amount of the nucleic acid target in the sample comprises determining a relative amount of the nucleic acid target. 
     
     
         30 . The method of  claim 27 , wherein quantifying the amount of the nucleic acid target in the sample comprises using end-point quantitation. 
     
     
         31 . The method of  claim 27 , wherein quantifying the amount of the nucleic acid target in the sample comprises determining an amount of the nucleic acid target by relating the PCR cycle number at which the signal is detectable over background to the amount of target present. 
     
     
         32 . The method of  claim 1 , further comprising detecting a signal from the reporter on the solid support prior to cleaving the hybridized target-specific probe. 
     
     
         33 . The method of  claim 1 , further comprising a linker between the target-specific probe and the solid support. 
     
     
         34 .- 99 . (canceled) 
     
     
         100 . A multiplex method for detecting the presence or absence of a plurality of target nucleic acids in a sample, comprising:
 (a) contacting the sample with a plurality of primer/probe pairs, each primer/probe pair comprising a target-specific primer complementary to a first region on a first strand of one of the plurality of target nucleic acids, and a target-specific probe complementary to a second region on the first strand of one of the plurality of target nucleic acids downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the first target-specific primer and the target-specific probe, wherein the target-specific probe comprises a reporter and is attached to a solid support;   (b) cleaving the hybridized target-specific probes with a nucleic acid polymerase having exonuclease activity to release the reporters from the solid support; and   (c) detecting a signal from the reporters on the solid support, whereby a change in the signal indicates the presence of a target nucleic acid.   
     
     
         101 . The method of  claim 100 , wherein the solid support is an encoded bead. 
     
     
         102 . The method of  claim 100 , wherein the reporter is a fluorphore. 
     
     
         103 . The method of  claim 102 , wherein the change in the signal is a decrease in a fluorescent signal. 
     
     
         104 . The method of  claim 3 , wherein the reporter is a fluorphore and quencher pair. 
     
     
         105 . The method of  claim 104 , wherein the change in the signal is an increase in a fluorescent signal. 
     
     
         106 . The method of  claim 100 , wherein each different target-specific probe of the plurality of primer/probe pairs is attached to a spatially discrete location on one solid. 
     
     
         107 . The method of  claim 100 , wherein each different target-specific probe of the plurality of (primer/probe pairs is attached to a different solid support. 
     
     
         108 . The method of  claim 100 , further comprising detecting a reference signal from a reporter on a non-hybridizing probe attached to a solid support. 
     
     
         109 . The method of  claim 108 , wherein the non-hybridizing probe is attached to a spatially discrete location on the same solid support to which the target-specific probes are attached. 
     
     
         110 . The method of  claim 108 , wherein the non-hybridizing probe is attached to a different solid support than that to which the target-specific probes are attached. 
     
     
         111 . The method of  claim 100 , wherein each different target-specific probe of the plurality of primer/probe pairs comprises the same reporter. 
     
     
         112 . The method of  claim 100 , wherein each different target-specific probe of the plurality of primer/probe pairs comprises a different reporter. 
     
     
         113 . The method of  claim 100 , further comprising contacting the sample with a plurality of different second target-specific primers complementary to a region on a second strand of the plurality of target nucleic acids, and performing multiple polymerase chain reaction cycles. 
     
     
         114 . The method of  claim 113 , wherein the multiple polymerase chain reaction cycles are performed without a wash step to remove free-floating reporters between cycles. 
     
     
         115 . The method of  claim 113 , wherein detecting the changes in signal from the reporters on the solid support comprises detecting the signals before and after performing the multiple polymerase chain reaction cycles. 
     
     
         116 . The method of  claim 113 , wherein detecting the changes in signals from the reporters on the solid support comprises detecting the signals only after performing the multiple polymerase chain reaction cycles. 
     
     
         117 . The method of  claim 113 , further comprising comparing the detected signals from the reporters on the solid support to a predetermined ratio of the signal of the reporter on the solid support to a reference signal from a reporter on a non-hybridizing probe attached to a solid support. 
     
     
         118 . The method of  claim 113 , further comprising quantifying the amount of the target nucleic acid in the sample.