Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor

Abstract

Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

What is claimed is: 
     
         1 . A set of chimeric oligonucleotide probes, the probe set comprising:
 a first chimeric oligonucleotide probe, comprising, in a 5′ to 3′ direction:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
 a target-specific portion, the target-specific portion having:
 complementarity to a 3′ portion of a preselected sequence of a target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; and 
 
   a second chimeric oligonucleotide probe comprising, in a 5′ to 3′ direction:
 a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 
 and 
 a primer-specific portion comprising an amplification primer nucleotide sequence; 
   wherein, when the first and second chimeric oligonucleotide probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the second chimeric oligonucleotide probe.   
     
     
         2 . The set of chimeric oligonucleotide probes of  claim 1 , wherein the target-specific portion of the second chimeric oligonucleotide probe comprises at least one nucleotide analog at one of the six 3′-most nucleotides, wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide. 
     
     
         3 . The set of chimeric oligonucleotide probes of  claim 2 , wherein the 3′-terminal and the 3′-penultimate nucleotides of the first chimeric oligonucleotide probe comprise non-modified ribonucleotides. 
     
     
         4 . The set of chimeric oligonucleotide probes of  claim 2 , wherein two, three, four, five, or six of the six 5′-most nucleotides of the target-specific portion of the first chimeric oligonucleotide probe comprise a nucleotide analog, and wherein two, three, four, five, or six of the six 3′-most nucleotides of the target-specific portion of the second chimeric oligonucleotide probe comprise a nucleotide analog, wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide. 
     
     
         5 . The set of chimeric oligonucleotide probes of  claim 4 , wherein the nucleotide analogs of the first chimeric oligonucleotide probe are contiguous. 
     
     
         6 . The set of chimeric oligonucleotide probes of  claim 4 , wherein the nucleotide sequence of the target-specific portion of the first chimeric oligonucleotide probe together with the nucleotide sequence of the target-specific portion of the second chimeric oligonucleotide probe is designed to anneal across an exon junction of the target nucleic acid. 
     
     
         7 . A set of chimeric oligonucleotide probes, the probe set comprising:
 at least two different species of first chimeric oligonucleotide probe wherein the 3′-terminal nucleotide of the species differ; and wherein each species of first chimeric oligonucleotide probe comprises:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
   a target-specific portion, the target-specific portion having:
 complementarity to a 3′ portion of a preselected sequence of a target nucleic acid for at least all but the 3′-terminal nucleotide, 
 a length of 6 nucleotides to 44 nucleotides, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; 
 a second chimeric oligonucleotide probe comprising, in a 5′ to 3′ direction: 
   a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, 
 complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 3′-most nucleotides, wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, 
   and   a primer-specific portion comprising an amplification primer nucleotide sequence;   wherein, when the two different species of first chimeric oligonucleotide probe and the second chimeric oligonucleotide probe are contacted with target nucleic acid under conditions suitable to allow annealing, the 5′ phosphate group of the second chimeric oligonucleotide probe is positioned immediately adjacent to the 3′ hydroxyl group of a species of first chimeric oligonucleotide probe having 3′-terminal nucleotide sequence complementarity to the target nucleic acid.   
     
     
         8 . A set of chimeric oligonucleotide probes, the probe set comprising:
 a first chimeric oligonucleotide probe, comprising, in a 5′ to 3′ direction:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
   a target-specific portion, the target-specific portion having:
 complementarity to a 3′ portion of a preselected sequence of a target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; 
   at least two different species of second chimeric oligonucleotide probe wherein the 5′-terminal nucleotide of the species differ; and wherein each species of second chimeric oligonucleotide probe comprises, in a 5′ to 3′ direction:
 a target-specific portion, the target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, complementarity to a 5′ portion of the preselected sequence of the target nucleic acid for at least all but the 5′-terminal nucleotide and, 
 a length of 6 nucleotides to 44 nucleotides, 
 and 
 a primer-specific portion comprising an amplification primer nucleotide sequence; 
 
   wherein, when the first chimeric oligonucleotide probe and the two different species of second chimeric oligonucleotide probe are contacted with target nucleic acid under conditions suitable to allow annealing, the 3′ hydroxyl group of the first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of a species of second chimeric oligonucleotide probe having 5′-terminal nucleotide sequence complementarity to the target nucleic acid.   
     
     
         9 - 12 . (canceled)