Probes for improved melt discrimination and multiplexing in nucleic acid assays

Patent metadata

Field	Value
Publication number	US-12480152-B2
Application number	US-202117196200-A
Country	US
Kind code	B2
Filing date	Mar 9, 2021
Priority date	Aug 11, 2014
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.

First claim

What is claimed is:

1 . A method for detecting the presence of a first target nucleic acid comprising:
(a) contacting the sample with a first set of probes, said set of probes comprising a first cleavable probe comprising, from 5′ to 3′, (i) a sequence region comprising at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a capture sequence; and (iii) a sequence comprising one or more ribonucleotide base(s) that is complementary to a first region on a first strand of the first target nucleic acid; and a first capture probe comprising, from 5′ to 3′, (i) a sequence region identical to the sequence region from the first cleavable probe and comprising at least one un-labeled non-natural nucleotide identical to the at least one non-natural nucleotide of the first cleavable probe; and (ii) a sequence complementary to the capture sequence of the first cleavable probe; (b) contacting the first cleavable probe with an endoribonuclease, thereby cleaving probe that is hybridized to the first target nucleic acid to form a first truncated probe; (c) allowing the first truncated probe to hybridize to the first capture probe; (d) extending the first truncated probe in the presence of a non-natural nucleotide that is labeled with a second member of the reporter-quencher pair and is capable of base-pairing with the at least one un-labeled non-natural nucleotide of the first capture probe to form a first extended probe; and (e) allowing the first extended probe to hybridize to itself to form a first hairpin probe; (f) detecting the first target nucleic acid by detecting a change in signal from the label on the first cleavable probe and the first hairpin probe.

2 . The method of claim 1 , wherein the endoribonuclease is RNase HII.

3 . The method of claim 1 , wherein detecting a change in signal from the label comprises detecting a change in signal from a reporter as the temperature of the sample is increased above the melt point of the first hairpin probe.

4 . The method of claim 1 , wherein the at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair or the non-natural nucleotide labeled with a second member of the reporter-quencher pair is isoG and the other is isoC.

5 . The method of claim 1 , further comprising:
(a) contacting the sample with a second set of probes, said second set of probes comprising a second cleavable probe comprising, from 5′ to 3′, (i) a sequence region comprising at least a one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a capture sequence; and (iii) a sequence comprising one or more ribonucleotide base(s) that is complementary to a first region on a first strand of a second target nucleic acid; and a second capture probe comprising, from 5′ to 3′, (i) a sequence region identical to the sequence region of the second cleavable probe and comprising at least one un-labeled non-natural nucleotide identical to the at least one non-natural nucleotide of the second cleavable probe; and (ii) a sequence complementary to capture sequence of the second cleavable probe; (b) contacting the second cleavable probe with an endoribonuclease, thereby cleaving probe that is hybridized to the second target nucleic acid to form a second truncated probe; (c) allowing the second truncated probe to hybridize to the second capture probe; (d) extending the second truncated probe in the presence of a non-natural nucleotide that is labeled with a second member of the reporter-quencher pair and is capable of base-pairing with the at least one un-labeled one non-natural nucleotide of the second capture probe to form a second extended probe; and (e) allowing the second extended probe to hybridize to itself to form a second hairpin probe; (f) detecting the second target nucleic acid by detecting a change in signal from the label on the second cleavable probe and the second hairpin probe.

6 . The method of claim 5 , wherein the first and second set of probes comprise distinguishable reporters.

7 . The method of claim 5 , wherein the first and second set of probes comprise the same reporter and wherein the first and second hairpin probes comprise distinguishable melt points.

8 . The method of claim 1 , further comprising performing multiple polymerase chain reaction cycles and wherein detecting the change in signal from the label comprises detecting the signal before and after performing the multiple polymerase chain reaction cycles.

9 . The method of claim 1 , further comprising performing multiple polymerase chain reaction cycles and wherein detecting the change in signal from the label comprises detecting the signal only after performing the multiple polymerase chain reaction cycles.

10 . The method of claim 9 , further comprising comparing the detected signal from the label to a predetermined ratio of the signal of the label relative to a reference signal from a label on a non-hybridizing probe.

11 . A method for detecting the presence of a first target nucleic acid comprising:
(a) contacting the sample with a first set of probes, said set of probes comprising a first cleavable probe comprising, from 5′ to 3′, (i) a sequence region comprising at least a one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a capture sequence; and (iii) a sequence comprising one or more ribonucleotide base(s) that is complementary to a first region on a first strand of the first target nucleic acid; and a first capture probe comprising, from 5′ to 3′, (i) a sequence region identical to a part of the sequence region of the first cleavable probe and (ii) a sequence complementary to the capture sequence of the first cleavable probe; (b) contacting the first cleavable probe with an endoribonuclease, thereby cleaving probe that is hybridized to the first target nucleic acid to form a first truncated probe; (c) allowing the first truncated probe to hybridize to the first capture probe; (d) extending the first truncated probe to form a first extended probe; (e) allowing the first extended probe to hybridize to itself to form a first hairpin probe; (f) further extending the first hairpin probe in the presence of a non-natural nucleotide that is labeled with a second member of the reporter-quencher pair and is capable of base-pairing with the at least one labeled non-natural nucleotide of the first cleavable probe; and (g) detecting the first target nucleic acid by detecting a change in signal from the label on the first cleavable probe and the first hairpin probe.

12 . The method of claim 11 , wherein the endoribonuclease is RNase HII.

13 . The method of claim 11 , 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 increased above the melt point of first hairpin probe.

14 . The method of claim 11 , wherein the at least one non-natural nucleotide labeled with a first member of the reporter-quencher pair or the non-natural nucleotide labeled with a second member of the reporter-quencher pair is isoG and the other is isoC.

15 . The method of claim 11 , further comprising:
(a) contacting the sample with a second set of probes, said set of probes comprising a second cleavable probe comprising, from 5′ to 3′, (i) a sequence region comprising at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a capture sequence; and (iii) a sequence comprising one or more ribonucleotide base(s) that is complementary to a first region on a first strand o

Assignees

• Luminex Corp

Inventors

• Johnson Scott C
• Arab Nicolas
• Whitman Doug

Classifications

• C12Q1/6823

  Release of bound markers · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q2527/101

  Temperature · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title