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1. (WO2005017173) PROCEDES ET COMPOSITIONS POUR AMPLIFIER DE L'ADN
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

We claim:

1. An Enzyme Blend for use with DNA comprising a DNA polymerase and a means for repairing an apurinic/apyrimidinic (AP) damage in DNA.

2. The Enzyme Blend of claim 1 , wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

3. The Enzyme Blend of claim 1, wherein the DNA has been damaged, is suspected of being damaged, or is undamaged.

4. The Enzyme Blend of claim 2, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI, REFl, APEX, Endonuclease IV, APNI, APEl (human endonuclease 1), or FEN-1.

5. The Enzyme Blend of claim 4, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.

6. The Enzyme Blend of claim 1 , further comprising a stabilizing agent.

7. The Enzyme Blend of claim 6, wherein the stabilizing agent is 1 ,4-dithioerythritol, DL-dithiothreitol, 2-mercaptoethanol, 2-mercaptoethanolamine, fericyanide, hydrazine, borane, or phosphine.

8. The Enzyme Blend of claim 1, further comprising a ligase.

9. The Enzyme Blend of claim 8, wherein the ligase is T4 DNA ligase.

10. The Enzyme Blend of claim 1, further comprising a DNA glycosylase.

11. The Enzyme Blend of claim 10, wherein the DNA glycosylase is uracil N-glycosylase.

12. The Enzyme Blend of claim 1, further comprising endonuclease IV.

13. The Enzyme Blend of claim 1 , further comprising DMSO .

14. The Enzyme Blend of claim 1 , further comprising a photolyase.

15. The Enzyme Blend of claim 14, wherein the photolyase is Thermus thermophilus photolyase.

16. The Enzyme Blend of claim 5 comprising:
a) 0.1 - 25 units/ul DNA polymerase; and
b) 5 - 50 units/ul AP endonuclease VI.

17. The Enzyme Blend of claim 16, further comprising:
a) 1-15 mM DTT; and
b) 10-50% v/v glycerol.

18. An Enzyme Blend comprising:
a) 2.5 units/ul DNA polymerase;
b) 5-50 units/ul AP endonuclease VI;
c) 10 mM Tris-HCl pH 8.0;
d) 150 mM KCl;
e) lOO ug/ml BSA;
f) 0.075 mM EDTA;
g) 7.5 mM DTT;
h) 0.25% v/v Tween 20;
i) 0.25% v/v IGEPAL CA-630; and
j) 50% v/v glycerol.

19. A kit comprising the Enzyme Blend of claim 1.

20. The kit of claim 19, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

21. The kit of claim 20, wherein the AP endonuclease DNA repair enzyme in the Enzyme Blend is AP endonuclease VI, REFl, APEX, Endonuclease IV, APNI, APEl (human endonuclease 1), or FEN-1.

22. The kit of claim 21 , wherein the AP endonuclease DNA repair enzyme is AP
endonuclease VI.

23. The kit of claim 22, wherein the Enzyme Blend comprises:
a) 0.1 - 25 units/ul DNA polymerase; and
b) 5 - 50 units/ul AP endonuclease VI.

24. The kit of claim 23, wherein the Enzyme Blend further comprises:
a) 1 - 15 mM DTT; and
b) 10 - 50% v/v glycerol.

25. A kit comprising the Enzyme Blend of claim 18.

26. A method for repairing DNA that is damaged or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of the Enzyme Blend of claim 1, and deoxynucleoside 5' triphosphates; and
b) incubating the mixture at 0°C - 99°C from about 0 sec. to about 3 hrs.

27. The method of claim 26, wherein the mixture is incubated for 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

28. The method of claim 26, wherein the DNA has a size of at least about 200 base pairs.

29. The method of claim 28, wherein the DNA has a size of at least about 500 base pairs.

30. The method of claim 26, wherein the DNA has a size of less than about 22,000 base pairs.

31. The method of claim 30, wherein the DNA has a size of less than about 1,000 base pairs.

32. The method of claim 26, wherein the DNA has a size of about 50 base pairs to about 500 base pairs.

33. The method of claim 26, wherein the DNA has a size of about 15,500 base pairs to about 22,000 base pairs.

34. The method of claim 26, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

35. A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of the Enzyme Blend of claim 1, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers,
wherein the pair of primers is substantially complementary to segments of the DNA; b) preincubating the mixture at 0°C - 99°C from about 0 sec. to about 3 hrs.;
c) denaturing the DNA; and
d) amplifying the DNA.

36. The method of claim 35, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

37. The method of claim 35, wherein step (d) is a polymerase chain reaction that comprises the steps of denaturation, annealing, and extension.

38. The method of claim 35, wherein step (d) is a rolling circle amplification.

39. The method of claim 35, wherein the pair of oligonucleotide primers have thiophosphate linkages.

40. The method of claim 39, wherein the thiophosphate linkages are located on the last two nucleotides at the 3' end of each oligonucleotide primer.

41. The method of claim 35, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

42. The method of claim 41, wherein the AP endonuclease DNA repair enzyme in the Enzyme Blend is AP endonuclease VI, REFl, APEX, Endonuclease IV, APNI, APEl (human endonuclease 1), or FEN-1.

43. The method of claim 42, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.

44. The method of claim 43, wherein the Enzyme Blend comprises:
a) 0.1 - 25 units/ul DNA polymerase; and
b) 5 - 50 units/ul AP endonuclease VI.

45. The method of 44, wherein the Enzyme Blend further comprises:
a) 3 - 15 mM DTT; and
b) 16 - 50% v/v glycerol.

46. The method of claim 35, where any or all of the steps are automated.

47. The method of claim 35, wherein the DNA has a size of at least about 200 base pairs.

48. The method of claim 47, wherein the DNA has a size of at least 500 base pairs.

49. The method of claim 35, wherein the DNA has a size of less than about 22,000 base pairs.

50. The method of claim 49, wherein the DNA has a size of less than about 1,000 base pairs.

51. The method of claim 35, wherein the damaged DNA has a size of about 50 base pairs to about 500 base pairs.

52. The method of claim 35, wherein the DNA has a size of about 15,500 base pairs to about 22,000 base pairs.

53. A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of the Enzyme Blend of claim 18, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers,
wherein the pair of primers is substantially complementary to segments of the DNA; b) preincubating the mixture at 0°C - 99°C from about 0 sec. to about 3 hrs.;
c) denaturing the DNA; and
d) amplifying the DNA.

54. The method of claim 53, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

55. A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of the Enzyme Blend of claim 1, and deoxynucleoside 5' triphosphates;
b) preincubating the mixture at a temperature of 0°C - 99°C from about 0 sec. to about 3 hrs.;

c) denaturing the DNA;
d) incubating the mixture at a temperature sufficient to inactivate an AP endonuclease
DNA repair enzyme in the Enzyme Blend and for a duration of time necessary to add a pair of oligonucleotide primers to the mixture, wherein the pair of primers is
substantially complementary to segments of the DNA;
e) adding the pair of oligonucleotide primers to the mixture; and
f) amplifying the DNA.

56. The method of claim 55, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

57. The method of claim 55, wherein step (f) is a polymerase chain reaction that comprises the steps of denaturation, annealing, and extension.

58. The method of claim 55, wherein step (f) is a rolling circle amplification.

59. The method of claim 55, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

60. The method of claim 59, wherein the AP endonuclease DNA repair enzyme in the Enzyme Blend is AP endonuclease VI, REFl, APEX, Endonuclease TV, APNI, APEl (human endonuclease 1), or FEN-1.

61. The method of claim 60, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.

62. The method of claim 61, wherein the Enzyme Blend comprises:
a) 0.1 - 25 units/ul DNA polymerase; and
b) 5 - 50 units/ul AP endonuclease VI.

63. The method of claim 62, wherein the Enzyme Blend further comprises : a) 3 - 15 mM DTT; and
b) 16 - 50% v/v glycerol.

64. The method of claim 55, wherein any or all of the steps are automated.

65. The method of claim 55, wherein the DNA has a size of at least about 200 base pairs.

66. The method of claim 65, wherein the DNA has a size of at least 500 base pairs.

67. The method of claim 55, wherein the DNA has a size of less than 22,000 base pairs.

68. The method of claim 67, wherein the DNA has a size of less than about 1,000 base pairs.

69. The method of claim 55, wherein the DNA has a size of about 50 base pairs to about 500 base pairs.

70. The method of claim 55, wherein the DNA has a size of about 15,500 base pairs to about 22,000 base pairs.

71. A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of the Enzyme Blend of claim 18, and deoxynucleoside 5' triphosphates;
b) preincubating the mixture at a temperature of 0°C - 99°C from about 0 sec. to about 3 hrs.;
c) denaturing the DNA;
d) incubating the mixture at a temperature sufficient to inactivate an AP endonuclease
DNA repair enzyme in the Enzyme Blend and for a duration of time necessary to add a pair of oligonucleotide primers to the mixture, wherein the pair of primers is
substantially complementary to segments of the DNA;
e) adding the pair of oligonucleotide primers to the mixture; and f) amplifying the DNA.

72. The method of claim 71, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

73. A method for preparation of an Enzyme Blend comprising combining a DNA polymerase with a means for repairing an AP damage in DNA in a vessel to form a blend.

74. The method of claim 73, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

75. The method of claim 74, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI, REFl, APEX, Endonuclease IV, APNI, APEl (human endonuclease 1), or

FEN-1.

76. The method of claim 75, wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.

77. The method of claim 71, wherein the Enzyme Blend further comprises a stabilizing agent.

78. The method of claim 77, wherein the stabilizing agent is 1,4-dithioerythritol, DL-dithiothreitol, 2-mercaptoethanol, 2-mercaptoethanolamine, fericyanide, hydrazine, borane, or phosphine.

79. The method of claim 71, wherein the Enzyme Blend further comprises a ligase.

80. The method of claim 79, wherein the ligase is T4 DNA ligase.

81. The method of claim 71 , wherein the Enzyme Blend further comprises a DNA
glycosylase.

82. The method of claim 81 , wherein the DNA glycosylase is Uracil N-glycosylase.

83. The method of claim 71, wherein the Enzyme Blend further comprises endonuclease IV.

84. The method of claim 71 , wherein the Enzyme Blend further comprises DMSO.

85. The method of claim 71, wherein the Enzyme Blend further comprises a photolyase.

86. The method of claim 85, wherein the photolyase is Thermus thermophilus photolyase.

87. A method for amplification of DNA that is damaged or suspected of being damaged comprising:
a) forming a mixture comprising the DNA, an effective amount of DNA polymerase, an effective amount of a means for repairing an AP damage in DNA, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers, wherein the pair of primers is
substantially complementary to segments of the DNA;
b) preincubating the mixture at 0°C - 99°C from about 0 sec. to about 3 hrs.;
c) denaturing the DNA; and
d) amplifying the DNA,
wherein the DNA has a size from about 50 base pairs to about 500 base pairs or has a size from about 15,500 base pairs to about 22,000 base pairs.

88. The method of claim 87, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

89. The method of claim 87, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

90. A method for rescue of DNA that is damaged or suspected of being damaged comprising: a) forming a mixture comprising the DNA, an effective amount of DNA polymerase, an effective amount of a means for repairing an AP damage in DNA, and
deoxynucleoside 5' triphosphates;
b) preincubating the mixture at a temperature of 0°C - 99°C from about 0 sec. to about 3 hrs.;
c) denaturing the DNA;
d) incubating of the mixture at a temperature sufficient to inactivate the AP
endonuclease DNA repair enzyme and for a duration of time necessary to add a pair of oligonucleotide primers to the mixture, wherein the pair of primers is substantially
complementary to segments of the DNA;
e) adding the pair of oligonucleotide primers to the mixture; and
f) amplifying the DNA, wherein the DNA has a size from about 50 base pairs to about 500 base pairs or has a size from about 15,500 base pairs to about 22,000 base pairs.

91. The method of claim 90, wherein the mixture is incubated at 0°C - 50°C from about 0 sec. to about 1 hr. in step (b).

92. The method of claim 90, wherein the means for repairing an AP damage in DNA is an AP endonuclease DNA repair enzyme.

93. An improved method for amplification of undamaged DNA comprising:
a) forming a mixture comprising the DNA, an effective amount of a DNA polymerase, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers having
thiophosphate linkages, wherein the pair of primers is substantially complementary to segments of the DNA;
b) denaturing the DNA; and
c) amplifying the DNA.

94. The method of claim 93, wherein step (c) is a polymerase chain reaction that comprises the steps of denaturation, annealing, and extension.

95. The method of claim 93, wherein step (c) is a rolling circle amplification.

96. The method of claim 93, wherein the thiophosphate linkages are located on the last two nucleotides at the 3' end of each oligonucleotide primer.

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