Probes for improved melt discrimination and multiplexing in nucleic acid assays

Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.

What is claimed is: 
     
         1 . A method for detecting the presence of a target nucleic acid comprising:
 (a) contacting the sample with a first cleavable probe, said probe comprising, from 5′ to 3′, (i) a first sequence region comprising at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence that is complementary to a first region on a first strand of the target nucleic acid;   (b) hybridizing the cleavable probe and an upstream primer to the target nucleic acid, and performing extension using a polymerase possessing 5′ nuclease activity;   (c) extending the upstream primer until contacting the cleavable probe with the polymerase possessing nuclease activity, thereby cleaving the probe that is hybridized to the target nucleic acid to form a truncated cleaved probe;   (d) allowing the truncated cleaved probe to hybridize to itself to form a hairpin probe;   (e) extending the hairpin probe in the presence of a non-natural nucleotide that is labeled with a second member of the reporter-quencher pair and is capable of base-pairing with the at least one non-natural nucleotide of the first sequence region; and   (f) detecting the target nucleic acid by detecting a change in signal from the label on the extended hairpin probe.   
     
     
         2 . The method of  claim 1 , further comprising performing melt analysis on the extended hairpin probe. 
     
     
         3 . The method of  claim 1  further including a loop sequence of one or more nucleotides between the second sequence region and the sequence that is the reverse complement of the second sequence region. 
     
     
         4 . The method of  claim 1  wherein the cleavable probe comprises an extension blocking modification at the 3′ end. 
     
     
         5 . The method of  claim 1  wherein detecting the presence of the target nucleic acid comprises detecting a change in signal from the reporter as the temperature is increased above the melt point of the extended hairpin probe. 
     
     
         6 . A method for detecting the presence of a target nucleic acid comprising:
 (a) contacting the sample with a first cleavable probe, said probe comprising, from 5′ to 3′, (i) a first sequence region labeled with a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence that is complementary to a first region on a first strand of the target nucleic acid;   (b) hybridizing the cleavable probe and an upstream primer to the target nucleic acid, and performing extension using a polymerase possessing 5′ nuclease activity;   (c) extending the upstream primer until contacting the cleavable probe with the polymerase possessing nuclease activity, thereby cleaving the probe that is hybridized to the target nucleic acid to form a truncated cleaved probe;   (d) allowing the truncated cleaved probe to hybridize to itself to form a hairpin probe;   (e) extending the hairpin probe using the first sequence region as a template to cause the flurophore and quencher to be physically separated; and   (f) detecting the target nucleic acid by detecting a change in signal from the extended hairpin probe.   
     
     
         7 . The method of  claim 6  further including a loop sequence of one or more nucleotides between the second sequence region and the sequence that is the reverse complement of the second sequence region. 
     
     
         8 . The method of  claim 6  wherein the cleavable probe comprises an extension blocking modification at the 3′ end. 
     
     
         9 . The method of  claim 6  wherein detecting the presence of the target nucleic acid comprises detecting a change in signal from the reporter as the temperature is increased above the melt point of the extended hairpin probe. 
     
     
         10 . The method of  claim 6  further comprising performing melt analysis on the extended hairpin probe.