Compositions and methods for enhancing hybridization and priming specificity
First Claim
1. A composition comprising a nucleic acid molecule and a salt, the salt comprising an anion and a cation, the anion selected from halogenated acetate, propionate and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms.
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Abstract
Compositions and methods are provided for increasing the specificity of a probe nucleic acid for a target nucleic acid in a hybridization solution. An abasic residue, deoxyNebularine residue, or a hybotrope is used to increase specificity. A method is provided for identifying useful hybotropes, including salts, water miscible organic solvents, aprotic solvents and organic solvents, on the basis of enthalpy considerations. Hybotropic hybridization and modified oligonucleotides may be used in amplification reactions, such as PCR, sequence analysis methods, and genomic screening methods.
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Citations
97 Claims
- 1. A composition comprising a nucleic acid molecule and a salt, the salt comprising an anion and a cation, the anion selected from halogenated acetate, propionate and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms.
- 19. A composition which is non-flowing comprising a nucleic acid molecule of 6-100 nucleotides and a salt, the salt comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate, and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbons.
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20. A composition which is free from organic solvent, comprising a nucleic acid molecule of 6-100 nucleotides and a salt, the salt comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate, and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbons.
- 29. A composition comprising a nucleic acid and a salt, the nucleic acid immobilized on a solid support, the salt comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbons.
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40. A salt selected from the group consisting of:
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(a) an acetate salt of a cation of the formula HN(CH3)2Ra wherein the nitrogen is positively charged and Ra is a C4-C7hydrocarbyl;
(b) a halogenated acetate salt of a cation of the formula HN(CH3)2Rb wherein the nitrogen is positively charged and Rb is a C7-C12hydrocarbyl;
(c) acetate and halogenated acetate salts of a cation of the formula H2N(C5-C7cycloalkyl)Rc where the nitrogen is positively charged and Rc is a C1-C12hydrocarbyl;
(d) acetate and halogenated acetate salts of N-substituted piperidine, wherein the nitrogen of piperidine is positively charged and substituted with C1-C12hydrocarbyl. - View Dependent Claims (41, 42, 43, 44, 45)
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46. An oligonucleotide in solution comprising a plurality of fragments, each fragment shown schematically by structure (1)
wherein - represents a sequence of at least three nucleotides as found in wild-type DNA, where “
B”
represents a base independently selected at each location;
represents a series of covalent chemical bonds termed a “
specificity spacer,”
which separates and connects two bases B3 and B5, wherein all nearest specificity spacers are separated by 8-12 nucleotides having a wild-type sequence;
the specificity spacer having steric and chemical properties such that (a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2)
and(b) it cannot enter into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2). - View Dependent Claims (47, 48, 49, 50)
wherein Y is selected from oxygen, sulfur, methyl and amino when X is oxygen, or Y is selected from oxygen and sulfur when X is sulfur; - and
SSC represents a specificity spacer component having a chain of 2-5 carbons shown in the formula wherein n is 0, 1, 2 or 3, and each of the shown 2-5 carbons of the specificity spacer component may be independently substituted with C1-C10hydrocarbyl or C1-C10hydrocarbyloxy, and any two of the shown 2-5 carbon atoms which are bonded directly to one another may form a carbocyclic or heterocyclic 5-6 membered ring.
- represents a sequence of at least three nucleotides as found in wild-type DNA, where “
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48. The oligonucleotide of claim 46 wherein n of the specificity spacer component is 1, and the specificity spacer component has the formula (2)
wherein n is 1 and X is selected from carbon, oxygen and sulfur, such that any carbon shown in formula (2), including X when it is carbon, may be substituted with hydrogen, C1-C5hydrocarbyl, C1-C5hydrocarbyloxy, a non-hydrogen bonding purine base analog or a non-hydrogen bonding pyrimidine base analog. -
49. The oligonucleotide of claim 47 wherein the specificity spacer component has the formula (3)
wherein each of the three shown carbons may be substituted with hydrogen, C1-C10hydrocarbyl or C1-C10hydrocarbyloxy. -
50. The oligonucleotide of claim 47 having a plurality of specificity spacers, where specificity spacers constitute 15-60% of the positions occupied by specificity spacers and nucleotides having a wild-type sequence.
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51. An array comprising a plurality of oligonucleotides immobilized in an array format to a solid support, each oligonucleotide of the plurality comprising a plurality of fragments, each fragment shown schematically by structure (1)
wherein, - represents a sequence of at least three nucleotides as found in wild-type DNA, where “
B”
represents a base independently selected at each location;
represents a series of covalent chemical bonds termed a “
specificity spacer,”
which separates and connects two bases B3 and B5, wherein all nearest specificity spacers are separated by 8-12 nucleotides having a wild-type sequence;
the specificity spacer having steric and chemical properties such that (a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2)
and(b) it cannot enter into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2). - View Dependent Claims (52, 53, 54, 55)
wherein Y is selected from oxygen, sulfur, methyl and amino when X is oxygen, or Y is selected from oxygen and sulfur when X is sulfur; - and
SSC represents a specificity spacer component having a chain of 2-5 carbon atoms shown in the formula wherein n is 0, 1, 2 or 3, and each of the shown 2-5 carbon atoms of the specificity spacer component may be independently substituted with C1-C10hydrocarbyl or C1-C10hydrocarbyloxy, and any two of the shown 2-5 carbon atoms which are bonded directly to one another may form a carbocyclic or heterocyclic 5-6 membered ring.
- represents a sequence of at least three nucleotides as found in wild-type DNA, where “
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53. The array of claim 51 wherein n of the specificity spacer component is 1, and the specificity spacer component has the formula (2)
wherein n is 1 and X is selected from carbon, oxygen and sulfur, such that any carbon shown in formula (2), including X when it is carbon, may be substituted with hydrogen, C1-C5hydrocarbyl, C1-C5hydrocarbyloxy, a non-hydrogen bonding purine base analog or non-hydrogen bonding pyrimidine base analog. -
54. The array of claim 51 wherein the specificity spacer component has the formula (3)
wherein each of the three shown carbons may be substituted with hydrogen, C1-C10hydrocarbyl or C1-C10hydrocarbyloxy. -
55. The array of claim 51 wherein each of the plurality of oligonucleotides have a plurality of specificity spacers, where specificity spacers constitute 15-60% of the positions occupied by specificity spacers and nucleotides having wild-type sequence.
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56. An oligonucleotide in solution comprising a plurality of fragments, each fragment shown schematically by structure (1)
wherein, - represents a sequence of at least three nucleotides as found in wild-type DNA, where “
B”
represents a base independently selected at each location;
represents a series of covalent chemical bonds termed a “
specificity spacer,”
which separates and connects two bases B3 and B5, wherein all nearest specificity spacers are separated by 8-12 nucleotides having a wild-type sequence;
the specificity spacer having steric and chemical properties such that (a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) (b) it enters into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2); and
(c) it does not hydrogen-bond through any of adenine, guanine, cytosine, thymine or uracil. - View Dependent Claims (57, 58, 59, 60)
wherein Y is selected from oxygen, sulfur, methyl and amino when X is oxygen, or Y is selected from oxygen and sulfur when X is sulfur; - and
SSC represents a specificity spacer component having a chain of 2-5 carbon atoms shown in the formula wherein n is 0, 1, 2 or 3, and each of the shown 2-5 carbon atoms of the specificity spacer component may be independently substituted with C1-C10hydrocarbyl or C1-C10hydrocarbyloxy, and any two of the shown 2-5 carbon atoms which are bonded directly to one another may form a carbocyclic or heterocyclic 5-6 membered ring.
- represents a sequence of at least three nucleotides as found in wild-type DNA, where “
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58. The oligonucleotide of claim 57 wherein n of the specificity spacer component is 1, and the specificity spacer component has the formula (2)
wherein n is 1 and X is selected from carbon, oxygen and sulfur, such that any carbon shown in formula (2), including X when it is carbon, may be substituted with hydrogen, C1-C5hydrocarbyl, C1-C5hydrocarbyloxy, a purine base analog or a pyrimidine base analog, where the purine base analog and the pyrimidine base analog may hydrogen bond to a complementary strand. -
59. The oligonucleotide of claim 57 wherein the specificity spacer component has the formula (3)
wherein each of the three shown carbon atoms may be substituted with hydrogen, C1-C10hydrocarbyl or C1-C10hydrocarbyloxy. -
60. The oligonucleotide of claim 56 having a plurality of specificity spacers, where specificity spacers constitute 15-60% of the positions occupied by specificity spacers and wild-type nucleotides.
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61. An array comprising a plurality of oligonucleotides immobilized in an array format to a solid support, each oligonucleotide of the plurality comprising a plurality of fragments, each fragment shown schematically by structure (1)
wherein, - represents a sequence of at least three nucleotides as found in wild-type DNA, where “
B”
represents a base independently selected at each location;
represents a series of covalent chemical bonds termed a “
specificity spacer,”
which separates and connects two bases B3 and B5, wherein all nearest specificity spacers are separated by 8-12 nucleotides having a wild-type sequence;
the specificity spacer having steric and chemical properties such that (a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) (b) it enters into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2); and
(c) it does not hydrogen-bond through any of adenine, guanine, cytosine, thymine or uracil. - View Dependent Claims (62, 63, 64, 65)
wherein Y is selected from oxygen, sulfur, methyl and amino when X is oxygen, or Y is selected from oxygen and sulfur when X is sulfur; - and
SSC represents a specificity spacer component having a chain of 2-5 carbon atoms shown in the formula wherein n is 0, 1, 2 or 3, and each of the shown 2-5 carbon atoms of the specificity spacer component may be independently substituted with C1-C10hydrocarbyl or C1-C10hydrocarbyloxy, and any two of the shown 2-5 carbon atoms which are bonded directly to one another may form a carbocyclic or heterocyclic 5-6 membered ring.
- represents a sequence of at least three nucleotides as found in wild-type DNA, where “
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63. The array of claim 62 wherein n of the specificity spacer component is 1, and the specificity spacer component has the formula (2)
wherein n is 1 and X is selected from carbon, oxygen and sulfur, such that any carbon shown in formula (2), including X when it is carbon, may be substituted with hydrogen, C1-C5hydrocarbyl, C1-C5hydrocarbyloxy, a purine base, a pyrimidine base, a non-hydrogen bonding purine base analog or a non-hydrogen bonding pyrimidine base. -
64. The array of claim 62 wherein the specificity spacer component has the formula (3)
wherein each of the three shown carbon atoms may be substituted with hydrogen, C1-C10hydrocarbyl or C1-C10hydrocarbyloxy. -
65. The array of claim 61 wherein each of the plurality of oligonucleotides have a plurality of specificity spacers, where specificity spacers constitute 15-60% of the positions occupied by specificity spacers and nucleotides having wild-type sequence.
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66. A method of distinguishing between hybridizations of a complementary nucleic acid target and a nucleic acid probe in which the probe and target are perfectly complementary and in which the probe and target have one or more base mismatches, comprising:
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(a) mixing the nucleic acid target with the nucleic acid probe in a solution comprising a hybotrope, the hybotrope comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate and halogenated propionate, and the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms;
(b) hybridizing at a discriminating temperature; and
(c) detecting hybridized probe and target, thereby determining whether the nucleic acid probe and target are perfectly complementary or mismatched. - View Dependent Claims (67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A method of distinguishing between hybridizations of a complementary nucleic acid target and a nucleic acid probe in which the probe and target are perfectly complementary and in which the probe and target have one or more base mismatches, comprising:
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(a) mixing a nucleic acid target with a nucleic acid probe containing at least one abasic residue or base analog in the presence of a hybotrope, the hybotrope present at a molarity of from about O.5M to about 6M;
(b) hybridizing at a discriminating temperature; and
(c) detecting hybridized probe and target, thereby determining whether the nucleic acid probe and target are perfectly complementary or mismatched. - View Dependent Claims (77, 78, 79, 80, 81, 82, 83, 84)
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85. A method of increasing discrimination in a nucleic acid synthesis procedure, comprising:
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(a) mixing a single-stranded nucleic acid target with an oligonucleotide primer in a solution comprising a hybotrope and a polymerase, where the hybotrope is present at a molarity of from about 0.5 M to about 6 M;
(b) annealing the primer to the target at a discriminating temperature; and
(c) synthesizing a complementary strand to the target to form a duplex. - View Dependent Claims (86, 87, 88, 89, 90, 91, 92)
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93. A method of distinguishing a single base change in a nucleic acid molecule from a wild-type sequence, comprising:
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(a) mixing a single-stranded nucleic acid target with an oligonucleotide primer in a solution comprising a hybotrope and a polymerase, where the hybotrope is present at a molarity of from about 0.5 M to about 6 M, and wherein the oligonucleotide primer has a 3′
-most base complementary to the wild-type sequence or the single base change;
(b) annealing the primer to the target at a discriminating temperature;
(c) extending the primer, wherein a complementary strand to the target is synthesized when the 3′
-most base of the primer is complementary to the target; and
(d) detecting the extension of the primer. - View Dependent Claims (94, 95, 96, 97)
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Specification