Detecting mutations in DNA

Patent metadata

Field	Value
Publication number	US-9637790-B2
Application number	US-201113991022-A
Country	US
Kind code	B2
Filing date	Dec 1, 2011
Priority date	Dec 3, 2010
Publication date	May 2, 2017
Grant date	May 2, 2017

Abstract

Provided herein are methods for detecting mutations in nucleic acid, and compositions and kits for performing such methods. In particular, nucleic acid amplification and fluorescence detection methods are provided to detect mutations and assess the mutational load. The methods are based on a set of adjacently binding probes wherein one probe is labelled with a quencher and the other is a self-indicating probe labelled with fluorophore and quencher. The binding of the probes is analyzed by melting curve analysis.

First claim

We claim:

1. A method for detecting mutations in mitochondrial DNA (mtDNA), chloroplastic DNA (cpDNA), or plasmid DNA, comprising
a) providing:
i) a sample comprising mtDNA, cpDNA, and/or plasmid DNA, and
ii) detection reagents comprising at least one pair of primers configured to amplify a target region of said mtDNA, cpDNA or plasmid DNA, and at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent sequences in said target region, the signaling probe comprising a fluorescence-emitting fluorophore and the quencher probe comprising a non-fluorescent quencher, wherein said signaling probe does not emit fluorescence above background when not hybridized to its target sequence and wherein when both the signaling probe and the associated quencher probe and are hybridized to the adjacent sequences in the target region the quencher probe quenches a fluorescent signal emitted by the fluorescence-emitting fluorophore of the signaling probe,
wherein the at least two probe sets comprise at least one probe that is part of both of the two probe sets, said probe:
comprising a quencher on each end, each end interacting with a different signaling probe from the at least two probe sets when both of the at least two probe sets are hybridized to the target region, or
comprising a fluorophore on one end and a quencher on the other end, the fluorophore interacting with a quencher probe in one of the at least two probe sets and the quencher interacting with a signaling probe of the other of the at least two probe sets when both of the at least two probe sets are hybridized to the target region;

b) amplifying said target region of said mtDNA, cpDNA, or plasmid DNA with said primers by a non-symmetric amplification method;
c) detecting the fluorescence of said fluorescence-emitting dye from each detectably distinguishable probe set at a range of temperatures;
d) generating temperature-dependent fluorescence signatures for each fluorescence-emitting dye; and
e) analyzing said temperature-dependent fluorescence signatures to detect mutations in said mtDNA, cpDNA or plasmid DNA.

2. The method of claim 1 , wherein said fluorescence-emitting fluorophore and said non-fluorescent quenchers of each probe set are capable of interacting by FRET.

3. The method of claim 1 , wherein the degree of complementarity between the probes of said probe sets and their target sequence varies based on the number of mutations in said target sequence, and wherein said different degrees of complementarity result in different temperature-dependent fluorescent signatures generated by said probe set and said target sequences.

4. The method of claim 1 , wherein detecting mutations in mtDNA comprises detecting mutations in one or more of the HV2, CO2, and ND1 regions of mtDNA.

5. A reagent kit for detecting one or more mutations in a target region of mtDNA, cpDNA, or plasmid DNA comprising:
a) at least one pair of primers, wherein said primers are configured bind to and amplify said target region of mtDNA, cpDNA or plasmid DNA; and
b) at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent sequences in said target region, the signaling probe comprising a fluorescence-emitting fluorophore and the quencher probe comprising a non-fluorescent quencher, wherein said signaling probe does not emit fluorescence above background when not hybridized to its target sequence and wherein when both the signaling probe and the associated quencher probe and are hybridized to the adjacent sequences in the target region the quencher probe quenches a fluorescent signal emitted by the fluorescence-emitting fluorophore of the signaling probe,
wherein the at least two probe sets comprise at least one probe that is part of both of the two probe sets, said probe:
comprising a quencher on each end, each end interacting with a different signaling probe from the at least two probe sets when both of the at least two probe sets are hybridized to the target region, or
comprising a fluorophore on one end and a quencher on the other end, the fluorophore interacting with a quencher probe in one of the at least two probe sets and the quencher interacting with a signaling probe of the other of the at least two probe sets when both of the at least two probe sets are hybridized to the target region.

6. The reagent kit of claim 5 , wherein the melting temperature of the signaling probe in at least one of the at least two probe sets is higher than the melting temperature of the associated quencher probe in the same probe set.

7. The reagent kit of claim 5 , wherein said fluorescence-emitting fluorophore and said non-fluorescent quenchers of each probe set are capable of interacting by FRET.

8. The reagent kit of claim 5 , wherein said reagent kit comprises 5 or more probe sets.

9. The reagent kit of claim 5 , wherein the degree of complementarity between the probes of said probe sets and their target sequence varies based on the number of mutations in said target sequence.

10. The reagent kit of claim 5 , wherein the probes in said at least one detectably distinguishable probe set have melting temperatures with their target nucleic acid sequences below the annealing temperature of at least one primer of the amplification reaction.

11. The reagent kit of claim 5 , wherein one or more detectably distinguishable probe sets are configured to hybridize to the HV2 region, CO2 region, or ND1 region of mtDNA.

12. The reagent kit of claim 11 , wherein at least one of said detectably distinguishable probe sets are selected from one or more of:
(a) SEQ ID NO:10 and SEQ ID NO:11;
(b) SEQ ID NO:12 and SEQ ID NO:13;
(c) SEQ ID NO:14 and SEQ ID NO:15;
(d) SEQ ID NO:16 and SEQ ID NO:17;
(e) SEQ ID NO:18 and SEQ ID NO:19;
(f) SEQ ID NO:20 and SEQ ID NO:21;
(g) SEQ ID NO:22 and SEQ ID NO:23;
(h) SEQ ID NO:24 and SEQ ID NO:25;
(i) SEQ ID NO:26 and SEQ ID NO:27;
(j) SEQ ID NO:28 and SEQ ID NO:29;
(k) SEQ ID NO:30 and SEQ ID NO:31;
(l) SEQ ID NO:32 and SEQ ID NO:33;
(m) SEQ ID NO:34 and SEQ ID NO:35;
(n) SEQ ID NO:36 and SEQ ID NO:37;
(o) SEQ ID NO:38 and SEQ ID NO:39; and
(p) SEQ ID NO:40 and SEQ ID NO:41.

13. The reagent kit of claim 11 , wherein said one or more primer pairs comprise:
(a) SEQ ID NO.:1 and SEQ ID NO.:2;
(b) SEQ ID NO.:3 and SEQ ID NO.:4; and
(c) SEQ ID NO.:5 and SEQ ID NO.:6.

14. The reagent kit of claim 5 , further comprising one or more additional oligonucleotides configured to suppress mis-priming during amplification reactions.

15. A method for detecting not previously known mutations in a target nucleic acid sequence, comprising
a) providing:
i) a sample comprising target nucleic acid, and
ii) detection reagents comprising at least one pair of primers configured to amplify a target region of said target nucleic acid sequence, and at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent sequences in said target region, the signaling probe comprising a fluorescence-emitting fluorophore and the quencher probe comprising a non-fluorescent quencher, wherein said signaling probe does not emit fluorescence above background when not hybridized to its target sequence and wherein when both the signaling probe and the associated quencher probe and are hybridized to the adjacent sequences in the target region the quencher probe quenches a fluorescent signal emitted by the fluorescence-emitting fluorophore of the signaling probe,
wherein the at le

Assignees

• Osborne Adam E
• Wangh Lawrence J
• Rice John E
• Univ Brandeis

Inventors

• Osborne Adam E
• Wangh Lawrence J
• Rice John E

Classifications

• C12Q1/6818 Primary

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

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12Q2527/107

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