Probes for improved melt discrimination and multiplexing in nucleic acid assays

Patent metadata

Field	Value
Publication number	US-11261481-B2
Application number	US-202016804060-A
Country	US
Kind code	B2
Filing date	Feb 28, 2020
Priority date	Aug 9, 2013
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of primers or probes that comprise a non-natural nucleotide linked to a reporter. Target nucleic acids are detected by the polymerization of a complementary probe or primer that incorporated a cognate non-natural nucleotide linked to a quencher.

First claim

What is claimed is:

1. A composition comprising a first set of probes, said set of probes comprising an upstream probe comprising, from 5′ to 3′, (i) at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a tag sequence; and (iii) a sequence complementary to a first region on a first strand of the target nucleic acid; and a downstream probe comprising, from 5′ to 3′, (i) a mirrored tag sequence having the same sequence as the tag sequence of the upstream probe; and (ii) a sequence complementary to a second region on a first strand of the target nucleic acid downstream of the first region, wherein the upstream probe comprises a 3′ sequence of 3 or more bases complementary to the downstream probe such that when hybridized to the target nucleic acid the set of probes form a T-junction.

2. The composition of claim 1 , further comprising a second set of probes comprising an upstream probe comprising, from 5′ to 3′, (i) at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a tag sequence; and (iii) a sequence complementary to a first region on a first strand of a second target nucleic acid; and a downstream probe comprising, from 5′ to 3′, (i) a mirrored tag sequence having the same sequence as the tag sequence of the upstream probe; and (ii) a sequence complementary to a second region on a first strand of the second target nucleic acid downstream of the first region, wherein the upstream probe comprises a 3′ sequence of 3 or more bases complementary to the downstream probe such that when hybridized to the target nucleic acid the set of probes form a T-junction.

3. The composition of claim 2 , wherein the first and second set of probes comprise distinguishable labels or form hairpin probes having distinguishable melt points.

4. The composition of claim 2 , comprising at least four sets of probes.

5. The composition of claim 1 , further comprising a reporter-labeled or quencher-labeled non-natural nucleotide.

6. The composition of claims 1 , further comprising a polymerase, a reference probe or free nucleotides.

7. A kit comprising:
(a) a first set of probes, said set of probes comprising an upstream probe comprising, from 5′ to 3′, (i) at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a tag sequence; and (iii) a sequence complementary to a first region on a first strand of the target nucleic acid; and a downstream probe comprising, from 5′ to 3′, (i) a mirrored tag sequence having the same sequence as the tag sequence of the upstream probe; and (ii) a sequence complementary to a second region on a first strand of the target nucleic acid downstream of the first region, wherein the upstream probe comprises a 3′ sequence of 3 or more bases complementary to the downstream probe such that when hybridized to the target nucleic acid the set of probes form a T-junction; and
(b) a reporter-labeled or quencher-labeled non-natural nucleotide.

8. The kit of claim 7 , comprising at least four sets of probes.

9. The kit of claim 8 , wherein the sets of probes comprise distinguishable labels or form hairpin probes having distinguishable melt points.

10. The kit of claim 7 , further comprising a polymerase, a reference probe, free nucleotides, a reference sample or instructions for use of the kit.

Assignees

• Luminex Corp

Inventors

• Whitman Doug
• Arab Nicolas
• Collins Chuck

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q2527/107

  Temperature of melting, i.e. Tm · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q2563/107

  fluorescence · CPC title