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

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 C1-C3 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.

A set of at least three oligonucleotide probes, comprising:

• a 5′-most 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 C1-C3 alkyl;
• a 3′-most 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;
• and
• a medial probe consisting essentially of:
• a target-specific portion having:
• a 5′-terminal nucleotide comprising a 5′-phosphate group,
• complementarity to a portion of the preselected sequence of the target nucleic acid intermediate the 5′-most and 3′-most oligonucleotide probes when annealed to target nucleic acid,
• a length of 6 nucleotides to 44 nucleotides,
• primarily non-modified ribonucleotides wherein any 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 C1-C3 alkyl;
• wherein, when the 5′-most, medial, and 3′-most probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the 5′-most chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the medial probe, and the 3′ hydroxyl group of the medial probe is positioned immediately adjacent to the 5′ phosphate group of the 3′-most chimeric oligonucleotide probe.