Probes for improved melt discrimination and multiplexing in nucleic acid assays

Patent metadata

Field	Value
Publication number	US-9657332-B2
Application number	US-201414456239-A
Country	US
Kind code	B2
Filing date	Aug 11, 2014
Priority date	Aug 9, 2013
Publication date	May 23, 2017
Grant date	May 23, 2017

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. An in vitro method for detecting the presence of a target nucleic acid comprising:
(a) contacting the sample with 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;
(b) extending the upstream probe to synthesize sequences complementary to the mirrored tag sequence on the downstream probe to form an extended upstream probe;
(c) allowing the extended upstream probe to hybridize to itself to form a hairpin probe;
(d) extending the hairpin probe in the presence of a non-natural nucleotide labeled with a second member of a reporter-quencher pair that is capable of base-pairing with the at least one non-natural nucleotide in the upstream probe; and
(e) detecting the target nucleic acid by detecting a change in signal from the label on the upstream probe and the hairpin probe.

2. The method of claim 1 , wherein the upstream probe comprises a 3′ sequence of 3 to 10 bases complementary to the tag sequence of the downstream probe.

3. The method of claim 1 , wherein the downstream probe further comprises (iii) a isoprimer complement sequence including one or more non-natural bases between the mirrored tag sequence having the same sequence as the tag sequence of the upstream probe and the sequence complementary to a second region on a first strand of the target nucleic acid downstream of the first region.

4. The method of claim 1 , wherein before being extended the upstream and downstream probes have a melt point of less than 55° C. in the absence of the target nucleic acid.

5. The method of claim 1 , wherein detecting a change in signal from the label comprises detecting a change in signal from the reporter as the temperature of the sample is changed.

6. The method of claim 1 , wherein the downstream probe is attached a solid support.

7. The method of claim 1 , wherein the at least one non-natural nucleotide or the quencher-labeled non-natural nucleotide is isoG and the other is isoC.

8. The method of claim 1 , further comprising:
(a) contacting the sample with a second, third, fourth, fifth or sixth set of probes, each 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, third, fourth, fifth or sixth 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, third, fourth, fifth or sixth 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;
(b) extending the upstream probe to synthesize sequences complementary to the mirrored tag sequence on the downstream probe to form an extended upstream probe;
(c) allowing the extended upstream probe to hybridize to itself to form a hairpin probe;
(d) extending the hairpin probe in the presence of a non-natural nucleotide labeled with a second member of a reporter-quencher pair that is capable of base-pairing with the at least one non-natural nucleotide in the upstream probe; and
(e) detecting the second, third, fourth, fifth or sixth target nucleic acid by detecting a change in signal from the label on the upstream probe and the hairpin probe.

9. The method of claim 1 , wherein the hairpin probes formed by the first and second set of probes comprise distinguishable melt points.

10. The method of claim 8 , wherein the hairpin probes formed by the first, second, third, fourth, fifth and sixth set of probes each comprise a distinguishable labels or distinguishable melt points.

11. An in vitro method for detecting the presence of one or both of a first and second target nucleic acid molecule in a sample comprising:
(a) contacting the sample with at least a first and second set of primers, each set of primers comprising a first primer comprising, from 5′ to 3′, (i) a reporter, (ii) a hairpin with a known melt point, (iii) a polymerase extension-blocking modification, (iv) at least one non-natural nucleotide, and (v) a target-specific sequence, complementary to a first region on the first strand of a target nucleic acid; and a second primer comprising a sequence complementary to a region on a second strand of the target nucleic acid, wherein the reporters from the first and second set of primer are the same and wherein the first and second set of primers comprise hairpins with distinguishable melt points;
(b) amplifying the target nucleic acids with the first and second set of primers, wherein said amplifying is performed in the presence of a quencher-labeled non-natural nucleotide that base-pairs with the non-natural nucleotide of the first primer of the first and second primer sets;
(c) detecting the presence of a target nucleic acid molecule by detecting a quenching of signal from the reporter; and
(d) determining whether the quenching of signal from the reporter resulted from the presence of the first target nucleic acid molecule, the second target nucleic acid molecule, or both by changing the temperature of the sample and determining the melt point or melt peaks corresponding to unquenching of the reporter, thereby detecting the presence of one or both of the first and second target nucleic acid molecules.

12. The method of claim 11 , wherein the reporter is a fluorophore.

13. The method of claim 12 , wherein the quenching of the signal is a decrease in a fluorescent signal.

14. The method of claim 11 , wherein one of the at least one non-natural nucleotide and the quencher-labeled non-natural nucleotide is isoG and the other is isoC.

15. The method of claim 11 , wherein the first and second set of primers comprise a first primer having hairpins with melt points that differ by between 2° C. and 10° C.

16. The method of claim 11 , further comprising:
(a) contacting the sample with at least a third, fourth, fifth or sixth set of primers, each set of primers comprising a first primer comprising, from 5′ to 3′, (i) a reporter, (ii) a hairpin with a known melt point, (iii) a polymerase extension-blocking modification, (iv) at least one non-natural nucleotide, and (v) a target-specific sequence, complementary to a first region on the first strand of a third, fourth, fifth or sixth target nucleic acid; and a second primer comprising a sequence complementary to a region on a second strand of the third, fourth, fifth or sixth target nucleic acid;
(b) amplifying a third, fourth, fifth or sixth target nucleic acid with the third, fourth, fifth or sixth set of primers, wherein said amplifying is performed in the presence of a quenche

Assignees

• Luminex Corp

Inventors

• Whitman Doug
• Arab Nicolas
• Collins Chuck

Classifications

• C12Q1/6818 Primary

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

• C12Q2561/109

  Invader technology · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2527/107

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

• C12Q2563/107

  fluorescence · CPC title