REAGENTS AND METHODS FOR ISOTHERMAL CHAIN REACTION

• US 20170335378A1
• Filed: 10/22/2015
• Published: 11/23/2017
• Est. Priority Date: 10/23/2014
• Status: Active Grant

• Current Assignee: Ricardo Mancebo
• Original Assignee: Ricardo Mancebo
• Inventors: Mancebo, Ricardo
• Granted Patent: US 10,612,077 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2523/109 chemical ligation between nucleic acids
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

In certain aspects, the invention disclosed herein relates to the isothermal amplification of probe linkage products to generate specific amplified signals. In some aspects, the invention provides methods, reagents, and kits for carrying out such amplification via the isothermal chain reaction (ICR).

Claims

1-45. -45. (canceled)

46. A method of amplifying a linkage product, the method comprising:

(a) forming a reaction solution comprising a nucleic acid molecule comprising a target nucleic acid template sequence, a probe nucleic acid (“

probe”

), and a universal linker nucleic acid (“

UL”

), wherein the probe comprises a first reactive moiety (“

BFRM1”

) on each of its 5′ and

3′

termini and/or at internal nucleotides, and the universal linker comprises a second reactive moiety (“

BFRM2”

) on each of its 5′ and

3′

termini and/or at internal nucleotides, the first reactive moiety being capable of reacting with the second reactive moiety to form a bond, wherein the probe comprises a nucleic acid sequence comprising, in 5′

to 3′

order;

(i) a first self-complementary region (“

SR1”

);

(ii) a target-complementary region (“

TR”

) comprising a nucleic acid sequence complementary to the target nucleic acid template sequence; and

(iii) a second self-complementary region (“

SR2”

) comprising a nucleic acid sequence complementary to the first complementary region;

wherein, in the absence of the target nucleic acid template sequence, the first self-complementary region hybridizes with the second self-complementary region such that the probe acquires a stem-loop structure and wherein bond formation between the first reactive moieties and the second reactive moieties is inhibited by the stem-loop structure; and

(b) incubating the reaction solution at an incubation temperature such that;

(1) the target-complementary region of the probe hybridizes to the target nucleic acid template sequence with a first melting temperature that is higher than the incubation temperature, wherein hybridization of the target-complementary region to the target nucleic acid template sequence disrupts the stem-loop structure of the probe, thereby disinhibiting reaction of the first reactive moieties with the second reactive moieties; and

(2) the first reactive moieties of the hybridized probe form chemical bonds with the second reactive moieties of the linker to form a linkage product, wherein the linkage product has a melting temperature for the target nucleic acid template sequence that is lower than the incubation temperature such that the linkage product disassociates from the target nucleic acid template sequence at the incubation temperature.

47. The method of claim 46, wherein the reaction solution comprises a plurality of probes and a plurality of universal linkers, and wherein steps (b)(1) and (b)(2) are repeated for the plurality of probes and the plurality of universal linkers such that a plurality of linkage products are formed.

48. The method of claim 46, wherein the linkage product disassociates from the target nucleic acid template sequence by thermal denaturation.

49. The method of claim 46, wherein the reaction solution comprises one or more organic solvents, high pH, cross-linking reagents, chaotropic agents, disulfide bond reducers, oligo wedges, and/or low salt concentrations to promote dissociation of the linkage product from the target nucleic acid template sequence.

50. The method of claim 46, wherein the first reactive moiety is selected from an alkyne (for example a cyclooctyne group) or an alkene (for example a trans-cyclooctene group) and the second reactive moiety is selected from an azide or aromatic ring (for example a tetrazine group), or the first reactive moiety is selected from an azide or aromatic ring and the second reactive moiety is selected from an alkyne or alkene.

51. The method of claim 46, wherein the first reactive moiety is selected from a nucleophilic group and the second reactive moiety is selected from an electrophilic group, or the first reactive moiety is selected from an electrophilic group and the second reactive moiety is selected from a nucleophilic group.

52. The method of claim 46, wherein the first reactive moiety is a phosphate group in the presence of a condensing agent (for example 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-activated phosphate group) and the second reactive moiety is a hydroxyl group, or the first reactive moiety is a hydroxyl group and the second reactive moiety is a condensing agent (for example 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-activated phosphate group).

53. The method of claim 46, wherein the first or second reactive moiety is a phosphorodithioate, phosphorotrithioate, 2′

,3′

-cyclic phosphate, amino-deoxyribonucleoside, thiol, amine, amino, hydrazine, hydrazide, bromide, azide, thiophosphate, iodide, chloride, maleimide, dabsylate, disulfide, tosylate, alkyne, isothiocyanate, cyclooctyne, trans-cyclooctene, NHS ester, imidoester, PFP ester, alkyl azide, aryl azide, isocyanate, nitrophenyl mono- or di-ester, tetrazine, aldehyde, epoxy, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-activated phosphate, hydroxyl, serinol, octadiynyl, hexynyl, I-Linker, carboxylate, succinimidyl-6-hydrazino-nicotinamide, succinimidyl-4-formylbenzamide, propargyl, or boronic acid.

54. The method of claim 46, wherein the probe is conjugated to a detectable moiety and/or quenching moiety.

55. The method of claim 46, wherein the universal linker is conjugated to a detectable moiety and/or a quenching moiety.

56. The method of claim 54, wherein the detectable moiety or quenching moiety is selected from the detectable moieties listed in Table I.

57. The method of claim 54, wherein the formation of the linkage product results in the separation of a detectable moiety from a quenching moiety.

58. The method of claim 46, wherein step (b) is performed at an isothermal temperature, and/or at a temperature that fluctuates and changes such as during thermocycling, and/or in a temperature gradient.

59. The method of claim 46, wherein the reaction solution comprises an enzyme that ligates the probe nucleic acid to the universal linker nucleic acid to generate a linkage product.

60. The method of claim 46, wherein a plurality of distinct linkage products are amplified in a single reaction solution by adding to the reaction solution a plurality of distinct probes having target-complementary regions complementary to distinct target nucleic acid template sequences.

61. The method of claim 60, wherein the plurality of distinct probes are conjugated to distinct detectable moieties and the plurality of distinct linkage products is detected.

62. The method of claim 61, wherein the linkage products are detected by high resolution melt curve analysis.

63. A method of detecting a nucleic acid molecule comprising a target nucleic acid template sequence, the method comprising:

(a) forming a reaction solution comprising a nucleic acid molecule comprising a target nucleic acid template sequence, a probe nucleic acid (“

probe”

), and a universal linker nucleic acid (“

UL”

), wherein the probe comprises a first reactive moiety on each of its 5′ and

3′

termini and/or at internal nucleotides, and the universal linker comprises a second reactive moiety on each of its 5′ and

3′

termini and/or at internal nucleotides, the first reactive moiety being capable of reacting with the second reactive moiety to form a bond, wherein the probe comprises a nucleic acid sequence comprising, in 5′

to 3′

order;

(i) a first self-complementary region (“

SR1”

);

(ii) a target-complementary region (“

TR”

) comprising a nucleic acid sequence complementary to the target nucleic acid template sequence; and

(iii) a second self-complementary region (“

SR2”

) comprising a nucleic acid sequence complementary to the first complementary region;

wherein, in the absence of the target nucleic acid template sequence, the first self-complementary region hybridizes with the second self-complementary region such that the probe acquires a stem-loop structure and wherein bond formation between the first reactive moieties and the second reactive moieties is inhibited by the stem-loop structure; and

(b) incubating the reaction solution at an incubation temperature such that;

(1) the target-complementary region of the probe hybridizes to the target nucleic acid template sequence with a first melting temperature that is higher than the incubation temperature, wherein hybridization of the target-complementary region to the target nucleic acid template sequence disrupts the stem-loop structure of the probe, thereby disinhibiting reaction of the first reactive moieties with the second reactive moieties; and

(2) the first reactive moieties of the hybridized probe form chemical bonds with the second reactive moieties of the linker to form a linkage product, wherein the linkage product has a melting temperature for the target nucleic acid template sequence that is lower than the incubation temperature such that the linkage product disassociates from the target nucleic acid template sequence at the incubation temperature; and

(c) detecting the linkage product.

64. The method of claim 63, wherein step (c) comprises detecting the fluorescence by FRET.

65. The method of claim 63, wherein step (c) comprises detecting loss of a signal emitted by the detectable moiety.

66. The method of claim 63, wherein step (c) comprises detecting a signal emitted by the detectable moiety.

67. The method of claim 63, wherein the formation of the linkage product results in the separation of the detectable moiety from the quenching moiety and step (c) comprises detecting a signal emitted by the detectable moiety.

68. The method of claim 63, wherein step (c) comprises detecting the linkage product using a double-stranded nucleic acid binding dye.

69. A method of detecting a plurality of nucleic acid molecules in multiplex with a single dye or detectable group, the plurality of nucleic acid molecules comprising multiple target nucleic acid template sequences, the method comprising:

(a) forming a reaction solution comprising a plurality of nucleic acid molecules comprising a plurality of target nucleic acid template sequences, a plurality of probe nucleic acids (“

probes”

), a plurality of universal linker nucleic acids (“

ULs”

), and a plurality of detector nucleic acids (“

detectors”

), wherein the probes comprise a first reactive moiety on each of their 5′ and

3′

termini and/or at internal nucleotides, and the universal linkers comprise a second reactive moiety on each of their 5′ and

3′

termini and/or at internal nucleotides, the first reactive moiety being capable of reacting with the second reactive moiety to form a bond, wherein each probe comprises a nucleic acid sequence comprising, in 5′

to 3′

order;

(i) a single-stranded self-complementary region (“

ssSR”

);

(ii) a first self-complementary region (“

SR1”

);

(iii) a target-complementary region (“

TR”

) comprising a nucleic acid sequence complementary to the target nucleic acid template sequence; and

(iv) a second self-complementary region (“

SR2”

) comprising a nucleic acid sequence complementary to the first complementary region;

wherein, in the absence of target nucleic acid template sequences, the first self-complementary region in each probe hybridizes with the second self-complementary region such that each probe acquires a stem-loop structure and wherein bond formation between the first reactive moieties and the second reactive moieties is inhibited by the stem-loop structure; and

(b) incubating the reaction solution at an incubation temperature such that;

(1) the target-complementary region of a plurality of probes hybridizes to a plurality of target nucleic acid template sequences with a first melting temperature that is higher than the incubation temperature, wherein hybridization of the target-complementary region from a plurality of probes to the plurality of target nucleic acid template sequences disrupts the stem-loop structure of the probes, thereby disinhibiting reaction of the first reactive moieties with the second reactive moieties; and

(2) the first reactive moieties of the plurality of hybridized probes form chemical bonds with the second reactive moieties of the plurality of linkers to form a plurality of linkage products in the same reaction, wherein the plurality of linkage products have melting temperatures corresponding to their target nucleic acid template sequences, which are lower than the incubation temperature such that the plurality of linkage products disassociate from their corresponding target nucleic acid template sequences at the incubation temperature; and

(c) detecting the plurality of linkage products by double-stranded nucleic acid binding dyes, wherein a plurality of detector nucleic acids hybridize to nucleotides in the ssSR, SR1, SR2, and UL in the plurality of linkage products to form a plurality of double-stranded nucleic acid regions for a nucleic acid binding dye to intercalate and bind for simultaneous multiplex detection of multiple linkage products in the same reaction.

70. The method of claim 69, wherein the probe nucleic acid, universal nucleic acid, detector nucleic acid, and/or probe linkage product are immobilized onto a solid support substrate.

71. The method of claim 69, wherein the probe nucleic acid, universal nucleic acid, detector nucleic acid, and/or probe linkage product are arrayed onto a solid support substrate.

72. A reagent composition for amplifying a probe linkage product in the presence of a target nucleic acid template sequence, the reagent composition comprising a probe nucleic acid comprising a first reactive moiety on each of its 5′

and 3′

termini and/or at internal nucleotides, and a universal linker nucleic acid comprising a second reactive moiety on each of its 5′ and

3′

termini and/or at internal nucleotides.

73. A kit comprising the reagent composition of claim 72 and instructions directing the use of the reagent composition for the amplification of a probe linkage product.

74. A device for performing probe linkage product nucleic acid amplification, comprising:

(a) an automated thermal cycler capable of maintaining an isothermal temperature, heating or cooling in a gradient temperature, or alternately heating and cooling in cycles in at least one reaction vessel comprising the reagent composition of claim 72, wherein the cycler and/or reaction vessel comprises the reagent composition;

(b) an excitation source for optically exciting the sample and causing the sample to fluoresce; and

(c) a photodetector for detecting a fluorescent signal from the sample while the amplification reaction is in progress, which fluorescent signal is proportional to the amount of amplified nucleic acid in the reaction vessel.

75. A method for performing a probe linkage product nucleic acid amplification of a first target nucleic acid template sequence comprising:

(a) forming a reaction solution by mixing in a reaction vessel a double-stranded nucleic acid binding dye with a sample comprising a reagent composition of claim 72 and a nucleic acid molecule comprising a first target nucleic acid template sequence;

(b) incubating the reaction solution at a constant isothermal temperature or heating or cooling the reaction solution in a temperature gradient or heating and cooling the reaction solution cyclically to amplify a probe linkage product nucleic acid;

(c) detecting the fluorescence of the double-stranded nucleic acid binding dye by melting the amplified probe linkage product nucleic acid to generate a first melting curve;

(d) repeating steps (a) through (c) with at least a second target nucleic acid template sequence to generate at least a second melting curve; and

(e) comparing the first and the at least second melting curves to determine a difference in the nucleic acid composition of the first and the at least second nucleic acid sequences.

76. A device for performing probe linkage product nucleic acid amplification, comprising:

(c) at least one reaction vessel comprising the reagent composition of claim 72;

(d) an excitation source for optically exciting the sample and causing the sample to fluoresce;

(e) a photodetector for detecting temperature-dependent fluorescence levels from the sample; and

(f) a processor programmed to generate a melting curve of the amplification product contained within the reaction vessel.