US 8,008,010 B1Granted

Chimeric oligonucleotides for ligation-enhanced nucleic acid detection, methods and compositions therefor

AssigneeApplied Biosystems, LLC

InventorsR. Scott Kuersten, Brittan L. Pasloske

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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.

Claims13

Dependent claims

A method for detecting a target nucleic acid in a sample, comprising:

a. contacting the sample with at least one set of chimeric oligonucleotide probes for a time and under conditions suitable to form an annealed product, wherein the at least one set of chimeric oligonucleotide probes comprises at least one first chimeric oligonucleotide probe and at least one second chimeric oligonucleotide probe;
wherein the at least one first chimeric oligonucleotide probe comprises, 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 the 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;
wherein the at least one second chimeric oligonucleotide probe comprises, 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 at least one first and the at least one 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;
b. contacting the annealed product with a polypeptide having double-strand dependent ligase activity for a time and under conditions suitable to form a ligated product;
and
c. detecting the target nucleic acid in the sample by detecting the ligated product, or a surrogate thereof.

16 more elements

A method for detecting a target nucleic acid in a sample, comprising:

a. contacting the sample with at least one set of chimeric oligonucleotide probes for each target nucleic acid to be detected for a time and under conditions suitable to form an annealed product, wherein the at least one set of chimeric oligonucleotide probes comprises at least one first chimeric oligonucleotide probe and at least one second chimeric oligonucleotide probe;
wherein the at least one first chimeric oligonucleotide probe comprises, 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 the 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;
wherein the at least one second chimeric oligonucleotide probe comprises, 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, a gap of one or more nucleotides separates the 3′ hydroxyl group of the first chimeric oligonucleotide probe and the 5′ phosphate group of the second chimeric oligonucleotide probe;
b. contacting the annealed product with a polymerase and nucleotides for a time and under conditions suitable to fill the gap;
c. contacting the product of b with a polypeptide having double-strand dependent ligase activity for a time and under conditions suitable to form a ligated product;
and
d. detecting the target nucleic acid by detecting the ligated product, or a surrogate thereof.

17 more elements

A method for detecting a target nucleic acid in a sample, comprising:

a. contacting the sample with at least one set of chimeric oligonucleotide probes for a time and under conditions suitable to form an annealed product, wherein the at least one set of chimeric oligonucleotide probes comprises at least one first chimeric oligonucleotide probe and at least one second chimeric oligonucleotide probe;
wherein the at least one first chimeric oligonucleotide probe comprises, 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 the target nucleic acid,
a length of 6 nucleotides to 44 nucleotides,
nucleotide analogs at six of the six 5′-most nucleotides wherein each 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 the at least one second chimeric oligonucleotide probe comprises, 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,
nucleotide analogs at six of the six 3′-most nucleotides wherein each nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide,
a length of 6 nucleotides to 44 nucleotides, and
a primer-specific portion comprising an amplification primer nucleotide sequence;
wherein, when the at least one first and at least one second chimeric oligonucleotide probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the at least one first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the at least one second chimeric oligonucleotide probe, and
wherein the target-specific portion of the at least one first chimeric oligonucleotide probe together with the target-specific portion of the at least one second chimeric oligonucleotide probe has a length of 15 nucleotides to 25 nucleotides;
b. contacting the annealed product with T4 Rnl2 ligase or an enzymatically active mutant or variant thereof, and with a single-strand specific ribonuclease, for a time and under conditions suitable to form a ligated product;
and
c. detecting the target nucleic acid by detecting the ligated product, or a surrogate thereof.

18 more elements

Dependencies are inferred from the claim text.