Compositions and methods for enhancing hybridization and priming specificity

• US 6,361,940 B1
• Filed: 04/01/1998
• Issued: 03/26/2002
• Est. Priority Date: 09/24/1996
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Qiagen Genomics, Inc. (Qiagen NV)
• Inventors: Garrison, Lori K., Van Ness, Jeffrey, Tabone, John C.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/053,831
• Time in Patent Office: 1,455 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/25.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence

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.

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.

2. The composition of claim 1 wherein the anion is trichloroacetate.

3. The composition of claim 1 wherein the anion is trifluoroacetate.

4. The composition of claim 1 wherein the cation is formed from atoms selected from 2-20 carbons, 0-5 oxygens and 1-5 nitrogens.

5. The composition of claim 1 wherein the cation has the structure HN(R)₃ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and any two R groups may join together to form a cyclic structure with the nitrogen.

6. The composition of claim 5 wherein R is independently selected from the group consisting of C₁-C₁₂alkyl, C₃-C₁₂cycloalkyl and C₇-C₁₂arylalkyl.

7. The composition of claim 1 wherein the cation has the structure N(H)₂(R)₂ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and the two R groups may join together to form a cyclic structure with the nitrogen.

8. The composition of claim 7 wherein R is independently selected from the group consisting of C₁-C₁₂alkyl, C₃-C₁₂cycloalkyl and C₇-C₁₂arylalkyl.

9. The composition of claim 1 wherein the cation is selected from the group consisting of ethylbutylammonium, 1-methylimidizole, 1-methylpiperidine, 1-methylpyrrolidine, 3-methoxypropylamine, triethylamine, bis(2-methoxyethyl)amine, diallylamine, dibutylamine, diisobutylamine, N,N-dimethylaminobutane, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine, N,N-dimethylhexylamine, triethanolamine, 1-ethylpiperidine, dicyclohexylamine, diisopropylamine, dipropylamine, N,N-dimethylisopropylamine, N-ethylbutylamine, tripropylamine, 2-methoxyethylamine, and N,N-dimethyloctylamine, and the anion is selected from the group consisting of trichloroacetate and trifluoroacetate.

10. The composition of claim 1 wherein the nucleic acid molecule comprises 6-100 nucleotides.

11. The composition of claim 1 wherein the nucleic acid molecule is DNA.

12. The composition of claim 1 wherein the nucleic acid molecule is immobilized on a solid support.

13. The composition of claim 1 wherein the nucleic acid molecules are arranged in an array on a solid support.

14. The composition of claim 1 further comprising an enzyme selected from polymerase and ligase.

15. The composition of claim 1 further comprising water.

16. The composition of claim 15 wherein the salt is completely dissolved in the water at a concentration of from 50 mM to 6 M at room temperature.

17. The composition of claim 15 wherein the nucleic acid molecule is present at a concentration of from 10⁻

6 to 10<sup>−

18</sup> g/mL.

18. The composition of claim 15 further comprising at least one of a buffer, detergent and chelator.

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.

21. The composition of any of claims 19 or 20 wherein the anion is acetate.

22. The composition of claim 19 wherein the cation is formed from atoms selected from 2-20 carbons, 0-5 oxygens, and 1-5 nitrogens.

23. The composition of claim 19 wherein the cation has the structure HN(R)₃ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and any two R groups may join together to form a cyclic structure with the nitrogen.

24. The composition of claim 19 wherein the cation has the structure N(H)₂(R)₂ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and the two R groups may join together to form a cyclic structure with the nitrogen.

25. The composition of claim 19 wherein the nucleic acid molecule is DNA.

26. The composition of claim 19 wherein the nucleic acid molecules are arranged in an array on a solid support.

27. The composition of claim 19 further comprising water.

28. The composition of claim 27 wherein the salt is completely dissolved in the water at a concentration of from 50 mM to 6 M at room temperature.

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.

30. The composition of claim 29 wherein the anion is selected from acetate, trifluoroacetate and trichloroacetate.

31. The composition of claim 29 wherein the cation is formed from atoms selected from 2-20 carbons, 0-5 oxygens, and 1-5 nitrogens.

32. The composition of claim 29 wherein the cation has the structure HN(R)₃ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and any two R groups may join together to form a cyclic structure with the nitrogen.

33. The composition of claim 29 wherein the cation has the structure N(H)₂(R)₂ wherein the nitrogen is positively charged, R is a C₁-C₁₂hydrocarbyl and the two R groups may join together to form a cyclic structure with the nitrogen.

34. The composition of claim 29 wherein the cation is selected from the group consisting of ethylbutylammonium, 1-methylimidizole, 1-methylpiperidine, 1-methylpyrrolidine, 3-methoxypropylamine, triethylamine, bis(2-methoxyethyl)amine, diallylamine, dibutylamine, diisobutylamine, N,N-dimethylaminobutane, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine, N,N-dimethylhexylamine, triethanolamine, 1-ethylpiperidine, dicyclohexylamine, diisopropylamine, dipropylamine, N,N-dimethylisopropylamine, N-ethylbutylamine, tripropylamine, 2-methoxyethylamine, and N,N-dimethyloctylamine, and the anion is selected from the group consisting of trichloroacetate and trifluoroacetate.

35. The composition of claim 29 wherein the solid support is selected from materials having a planar surface and comprising quartz, gold, nylon-6,6, nylon, polystyrene, glass, and silicon.

36. The composition of claim 35 wherein the solid support is selected from a glass plate and a silicon wafer.

37. The composition of claim 29 wherein the nucleic acid molecules are arranged in separated domains in an array, where the number of domains present in an array is selected from the ranges 10 to 50, 50 to 400, and 400 to 800.

38. The composition of claim 37 wherein the domains are substantially circular, where the circles have an average diameter of about 10 microns to 200 microns.

39. The composition of claim 29 wherein the nucleic acids comprise a plurality of sequences.

40. A salt selected from the group consisting of:

(a) an acetate salt of a cation of the formula HN(CH₃)₂R_a wherein the nitrogen is positively charged and R^a is a C₄-C₇hydrocarbyl;

(b) a halogenated acetate salt of a cation of the formula HN(CH₃)₂R_b wherein the nitrogen is positively charged and R_b is a C₇-C₁₂hydrocarbyl;

(c) acetate and halogenated acetate salts of a cation of the formula H₂N(C₅-C₇cycloalkyl)R_c where the nitrogen is positively charged and R_c 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.

41. The salt of claim 40 wherein hydrocarbyl is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, and alkylaryl.

42. The salt of claim 40 which is an acetate salt of a cation of the formula HN(CH₃)₂R_a wherein the nitrogen is positively charged and R_a is a C₄-C₇hydrocarbyl.

43. The salt of claim 40 which is a halogenated acetate salt of a cation of the formula HN(CH₃)₂R_b wherein the nitrogen is positively charged and R_b is a C₇-C₁₂hydrocarbyl.

44. The salt of claim 40 which is an acetate or halogenated acetate salt of a cation of the formula H₂N(C₅-C₇cycloalkyl)R_c where the nitrogen is positively charged and R_c is a C₁-C₁₂hydrocarbyl.

45. The salt of claim 40 which is an acetate or halogenated acetate salt of N-substituted piperidine, wherein the nitrogen of piperidine is positively charged and substituted with C₁-C₁₂hydrocarbyl.

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).

47. The oligonucleotide of claim 46 wherein the specificity spacer has the formula

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.

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.

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).

52. The array of claim 51 wherein the specificity spacer has the formula

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.

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.

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.

57. The oligonucleotide of claim 56 wherein the specificity spacer has the formula

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.

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.

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.

62. The array of claim 61 wherein the specificity spacer has the formula

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.

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.

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:

(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.

67. The method according to claim 66 wherein the nucleic acid probe is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme.

68. The method according to claim 66 wherein the nucleic acid probe is from 6 to 40 bases.

69. The method according to claim 66 wherein the target nucleic acid probe is from 6 to 40 bases.

70. The method according to claim 66 wherein the hybotrope is selected from the group consisting of bis(2-methoxyethyl)amine acetate, 1-ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, and tripropylamine acetate.

71. The method according to claim 66 wherein the hybotrope is a compound that has a pK₁ of less than 2.2 in water at 25°

C.

72. The method according to claim 66 wherein the hybotrope is present at a molarity of from about 0.5 M to about 6 M.

73. The method according to claim 66 wherein the probe nucleic acid is DNA or RNA.

74. The method according to claim 66 wherein the target nucleic acid is genomic DNA, RNA, or cDNA.

75. The method according to claim 66 wherein the target nucleic acid is affixed to a solid substrate.

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:

(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.

77. The method according to claim 76 wherein the base analog is deoxyNebularine.

78. The method according to claim 76 wherein the nucleic acid probe is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme.

79. The method according to claim 76 wherein the nucleic acid probe is from 6 to 40 bases.

80. The method according to claim 76 wherein the target nucleic acid probe is from 6 to 40 bases.

81. The method according to claim 76 wherein the hybotrope is selected from the group consisting of bis(2-methoxyethyl)amine acetate, 1-ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate.

82. The method according to claim 76 wherein the probe nucleic acid is DNA or RNA.

83. The method according to claim 76 wherein the target nucleic acid is genomic DNA, RNA or cDNA.

84. The method according to claim 76 wherein the target nucleic acid is affixed to a solid substrate.

85. A method of increasing discrimination in a nucleic acid synthesis procedure, comprising:

(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.

86. The method according to claim 85 wherein the nucleic acid primer is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme.

87. The method according to claim 85 wherein the nucleic acid primer is from 6 to 40 bases.

88. The method according to claim 85 wherein the hybotrope is selected from the group consisting of bis(2-methoxyethyl)amine acetate, 1 -ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate.

89. The method according to claim 85 wherein the hybotrope is an amine-based salt selected from the group consisting of ethylpiperidine acetate, ethylbutylamine acetate, bismethoxyamine acetate, dipropylamine acetate and diisopropylamine acetate.

90. The method according to claim 85 wherein the hybotrope is ethyl piperidine.

91. The method according to claim 85 wherein the hybotrope is present at a molarity of from about 10 M to about 1 M.

92. The method according to claim 85 wherein the steps of (a), (b), and (c) are repeated multiple times.

93. A method of distinguishing a single base change in a nucleic acid molecule from a wild-type sequence, comprising:

(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.

94. The method of claim 93, further comprising in step (a) a second primer having a wild-type sequence, wherein the second primer anneals to the strand synthesized in step (c).

95. The method of claim 94 wherein steps (a), (b) and (c) are performed multiple times.

96. The method of claim 94 wherein the hybotrope is selected from the group consisting of bis(2-methoxyethyl)amine acetate, 1-ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate.

97. The method of claim 94 wherein the hybotrope is 1-ethylpiperidine acetate.