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1. (WO2015176006) PLATEFORME DE DÉTECTION GÉNÉTIQUE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

CLAIMS

WHAT IS CLAIMED IS:

1. A composition comprising a polymerase comprising at least one albumin binding

moiety.

2. The composition of claim 1, wherein the polymerase is a DNA polymerase or an R A polymerase.

3. The composition of claim 2, wherein the polymerase comprises Taq polymerase, Vent polymerase, Klenow Fragment (3 '-5' exo-), DNA Polymerase I (large Klenow fragment), E. coli DNA polymerase I, phi29 DNA polymerase, Phusion DNA

polymerase, or T4 DNA polymerase.

4. The composition of claim 3, wherein the polymerase is Taq polymerase.

5. The composition of claim 4, wherein the Taq polymerase is native or modified Taq

polymerase.

6. The composition of claim 4 or 5, wherein the Taq polymerase further comprises a HIS moiety, a biotin-tag moiety, Z domain moiety, or combinations thereof.

7. The composition of claim 6, wherein the at least one albumin binding moiety is directly connected to the Taq polymerase or is connected to the Taq polymerase though a spacer.

8. The composition of claim 6 or 7, wherein a genetic sequence of the at least one albumin binding moiety and the Taq polymerase comprises the at least one albumin binding moiety sequence residing on the 3 ' end of the Taq polymerase sequence, residing on the 5' end of the Taq polymerase sequence, or residing on both the 3' end and 5' end of the Taq polymerase sequence.

9. The composition of claim 1, wherein the composition further comprises an albumin.

10. The composition of claim 9, wherein the albumin inhibits the activity of the polymerase by binding to the polymerase at a temperature of from about 0 °C to about 60 °C, from about 20 °C to about 55 °C, or from about 25 °C to about 50 °C.

11. The composition of claim 9 or 10, wherein the albumin is inactivated at a temperature of at least 61 °C or higher.

12. The composition of any one of the claims 9-11, wherein the polymerase regains its

enzymatic activity at a temperature of at least 61 °C or higher.

13. The composition of any one of the claims 9-12, wherein the albumin inhibits the activity of the polymerase by about 10% to about 100% relative to a control.

14. The composition of claim 13, wherein the control is the activity of an equivalent

polymerase in the absence of a polymerase inhibitor.

15. The composition of any one of the claims 9-14, wherein the albumin is mammalian albumin or a mammalian albumin analogue.

16. The composition of any one of the claims 9-15, wherein the albumin is human serum albumin.

17. The composition of any one of the claims 9-15, wherein the albumin is bovine serum albumin.

18. The composition of any one of the claims 1-17, wherein the albumin binding moiety able to bind serum albumin is at least a part of Streptococcal protein G.

19. The composition of claim 18, wherein the at least a part of Streptococcal protein G is the entire Streptococcal protein G.

20. The composition of claim 18, wherein the at least a part of Streptococcal protein G

comprises ABP (121aa), BB (214aa), ABD (46aa), ADB1 binding site, ADB2 binding site, or ADB3 binding site.

21. The composition of claim 20, wherein the ABD to albumin affinity is 1.5 nanomolar or less.

22. The composition of claim 20, wherein the ABD to human serum albumin affinity is 1.5 nanomolar or less.

23. The composition of any one of the claims 1-22, wherein the polymerase has the sequence as illustrated in SEQ ID NO: 1.

24. The composition of any one of the claims 1-23, wherein the polymerase is expressed in an eukaryotic cell.

25. The composition of claim 24, wherein the eukaryotic cell is a yeast cell.

26. The composition of claim 25, wherein the yeast is Pichia pastoris.

27. The composition of any one of the claims 1-23, wherein the polymerase is expressed in E. coli.

28. The composition of any one of the claims 1-27, wherein the polymerase has less than about 30ng/mL of nucleic acid contaminant.

29. A reaction mixture comprising:

a) a polymerase comprising an albumin binding moiety; and

b) an albumin.

30. The reaction mixture of claim 29, wherein the albumin inhibits the activity of the

polymerase by binding to the polymerase at a temperature of from about 0 °C to about 60 °C, from about 20 °C to about 55 °C, or from about 25 °C to about 50 °C.

31. The reaction mixture of claim 29 or 30, wherein the albumin is released from the polymerase at a temperature of at least 61 °C or higher.

32. The reaction mixture of any one of the claims 29-31 , wherein the polymerase regains its enzymatic activity at a temperature of at least 61 °C or higher.

33. The reaction mixture of any one of the claims 29-32, wherein the albumin inhibits the activity of the polymerase by about 10% to about 100% relative to a control.

34. The reaction mixture of claim 33, wherein the control is the activity of an equivalent polymerase in the absence of a polymerase inhibitor.

35. The reaction mixture of any one of the claims 29-34, wherein the polymerase is a DNA polymerase or an RNA polymerase.

36. The reaction mixture of any one of the claims 29-35, wherein the polymerase comprises Taq polymerase, Vent polymerase, Klenow Fragment (3 '-5' exo-), DNA Polymerase I (large Klenow fragment), E. coli DNA polymerase I, phi29 DNA polymerase, Phusion DNA polymerase, or T4 DNA polymerase.

37. The reaction mixture of any one of the claims 29-36, wherein the polymerase is Taq polymerase.

38. The reaction mixture of claim 37, wherein the Taq polymerase is native or modified Taq polymerase.

39. The reaction mixture of claim 29, wherein the albumin binding moiety is directly

connected to the Taq polymerase or is connected to the Taq polymerase though a spacer.

40. The reaction mixture of claim 39, wherein a genetic sequence of the albumin binding moiety and the Taq polymerase comprises the albumin binding moiety sequence residing on the 3' end of the Taq polymerase sequence, residing on the 5' end of the Taq polymerase sequence, or residing on both the 3' end and 5' end of the Taq polymerase sequence.

41. The reaction mixture of any one of the claims 29-40, wherein the albumin is mammalian albumin or a mammalian albumin analogue.

42. The reaction mixture of any one of the claims 29-41 , wherein the albumin is human serum albumin.

43. The reaction mixture of any one of the claims 29-41 , wherein the albumin is bovine serum albumin.

44. The reaction mixture of any one of the claims 29-43, wherein the albumin binding

moiety able to bind serum albumin is at least a part of Streptococcal protein G.

45. The reaction mixture of claim 44, wherein the at least a part of Streptococcal protein G is the entire Streptococcal protein G.

46. The reaction mixture of claim 44, wherein the at least a part of Streptococcal protein G comprises ABP (121aa), BB (214aa), ABD (46aa), ADB1 binding site, ADB2 binding site, or ADB3 binding site.

47. The reaction mixture of claim 46, wherein the ABD to albumin affinity is 1.5 nanomolar or less.

48. The reaction mixture of claim 46, wherein the ABD to human serum albumin affinity is 1.5 nanomolar or less.

49. The reaction mixture of any one of the claims 29-48, wherein the polymerase further comprises a HIS moiety, a biotin-tag moiety, Z domain moiety, or a combination thereof.

50. The reaction mixture of any one of the claims 29-49, wherein the polymerase has the sequence as illustrated in SEQ ID NO: 1.

51. The reaction mixture of any one of the claims 29-50, wherein the reaction mixture is an amplification reaction mixture.

52. The reaction mixture of claim 51 , wherein the amplification is a polymerase chain

reaction (PCR).

53. The reaction mixture of claim 51 or 52, wherein the amplification comprises whole

genome amplification, helicase dependent amplification, nicking enzyme amplification reaction, reverse transcription PCR (RT-PCR), ligation mediated PCR, methylation specific PCR, digital PCR, hot start PCR, multiplex ligation-dependent probe

amplification (MLPA), multiplex -PCR, nested PCR, overlap-extension PCR, or quantitative PCR (qPCR).

54. The reaction mixture of claim 51 or 52, wherein the amplification is a next-generation sequencing method.

55. A polymerase construct comprising at least one moiety that is capable of binding

albumin.

56. The polymerase construct of claim 55, wherein the polymerase is a DNA polymerase or an RNA polymerase.

57. The polymerase construct of claim 55 or 56, wherein the polymerase comprises Taq

polymerase, Vent polymerase, Klenow Fragment (3 '-5' exo-), DNA Polymerase I (large Klenow fragment), E. coli DNA polymerase I, phi29 DNA polymerase, Phusion DNA polymerase, or T4 DNA polymerase.

58. The polymerase construct of claim 57, wherein the polymerase is Taq polymerase.

59. The polymerase construct of claim 58, wherein the Taq polymerase is native or modified Taq polymerase.

60. The polymerase construct of any one of the claims 55-59, wherein the at least one moiety is directly connected to the Taq polymerase or is connected to the Taq polymerase though a spacer.

61. The polymerase construct of any one of the claims 55-60, wherein a genetic sequence of the at least one moiety and the Taq polymerase comprises the at least one moiety sequence residing on the 3 ' end of the Taq polymerase sequence, residing on the 5 ' end of the Taq polymerase sequence, or residing on both the 3' end and 5' end of the Taq polymerase sequence.

62. The polymerase construct of any one of the claims 55-61, wherein the albumin is

mammalian albumin or a mammalian albumin analogue.

63. The polymerase construct of any one of the claims 55-62, wherein the albumin is human serum albumin or bovine serum albumin.

64. The polymerase construct of any one of the claims 55-63, wherein the at least one moiety bind to serum albumin.

65. The polymerase construct of any one of the claims 55-64, wherein the at least one moiety able to bind serum albumin is at least a part of Streptococcal protein G.

66. The polymerase construct of claim 65, wherein the at least a part of Streptococcal protein G is the entire Streptococcal protein G.

67. The polymerase construct of claim 65, wherein the at least a part of Streptococcal protein G comprises ABP (121aa), BB (214aa), ABD (46aa), ADB1 binding site, ADB2 binding site, or ADB3 binding site.

68. The polymerase construct of claim 67, wherein the ABD to albumin affinity is 1.5

nanomolar or less.

69. The polymerase construct of claim 67, wherein the ABD to human serum albumin

affinity is 1.5 nanomolar or less.

70. The polymerase construct of any one of the claims 55-69, wherein the polymerase

construct further comprises a HIS moiety, a biotin-tag moiety, Z domain moiety, or a combination thereof.

71. The polymerase construct of any one of the claims 55-70, wherein the polymerase

construct is the construct as illustrated in SEQ ID NO: 1.

72. A method for amplifying a target DNA comprising:

a) incubating the target DNA with a polymerase having the polymerase construct of claims 55-71, an albumin, a set of primers, and nucleoside phosphates selected from the group consisting of adenine, thymine, guanine, cytosine, and uridine; so as to form a reaction mixture; and

b) subjecting the reaction mixture to an amplification method, whereby the set of primers is extended by the polymerase to amplify the target DNA sequence.

73. The method of claim 72, wherein the albumin inhibits the activity of the polymerase by binding to the polymerase at a temperature of from about 0 °C to about 60 °C, from about 20 °C to about 55 °C, or from about 25 °C to about 50 °C.

74. The method of claim 72 or 73, wherein the albumin is inactivated at a temperature of at least 61 °C or higher.

75. The method of any one of the claims 72-74, wherein the polymerase regains its

enzymatic activity at a temperature of at least 61 °C or higher.

76. The method of claim 72, wherein the polymerase is expressed in an eukaryotic cell.

77. The method of claim 76, wherein the polymerase is expressed in Pichia pastoris.

78. The method of claim 72, wherein the amplification method is a polymerase chain

reaction (PCR).

79. An albumin affinity separation method for enzyme purification comprising:

a) forming a protein construct comprising a target polymerase bound to an albumin binding moiety;

b) contacting the protein construct with albumin to form an albumin molecular complex;

c) separating the protein construct; and

d) retrieving the protein construct from the albumin molecular complex, wherein the protein construct retains activity of the target polymerase.

80. The method of claim 79, wherein the protein construct further comprises a HIS binding moiety.

81. The method of claim 79 or 80, wherein the method further comprises purifying the

protein construct using a HIS affinity separation method.

82. The method of claim 81 , wherein the HIS affinity separation method comprises:

a) contacting the protein construct with HIS binding moiety to form a HIS molecular complex;

b) separating the protein construct from species not bound to HIS binding moiety; and

c) retrieving the protein construct from the HIS molecular complex, wherein the protein construct retains the activity of the target polymerase.

83. The method of claim 82, wherein the HIS affinity separation method precedes the

albumin affinity separation method, or the albumin affinity separation method precedes the His affinity separation method.

84. The method of claim 79, wherein the protein construct further comprises a biotin-tag moiety, a Z domain moiety, or a combination thereof.

85. The method of claim 79, wherein the retrieving comprises altering the pH of at least the environment immediately surrounding the albumin molecular complex.

86. The method of claim 85, wherein the altering the pH is elevating the pH value of at least the environment immediately surrounding the albumin molecular complex, or reducing the pH value of at least the environment immediately surrounding the albumin molecular complex.

87. The method of claim 79, wherein the retrieving comprises altering the salt concentration of at least the environment immediately surrounding the albumin molecular complex, altering the conductivity of at least the environment immediately surrounding the albumin molecular complex, altering the temperature of at least the environment immediately surrounding the albumin molecular complex, or a combination thereof.

88. The method of claim 79, wherein the separating comprises washing with an aqueous solution.

89. The method of claim 79, wherein the albumin is mammalian albumin or a mammalian albumin analogue.

90. The method of claim 79 or 89, wherein the albumin is human serum albumin or bovine serum albumin.

91. The method of any one of the claims 79-90 wherein the albumin is bound to a solid support or bound to particles.

92. The method of claim 91, wherein the particles are assembled into a column.

93. The method of claim 91 or 92, wherein the particles are magnetic particles.

94. The method of claim 91 , wherein the bond is covalently bound.

95. The method of claim 94, wherein the covalently bound is direct or indirect through a molecular spacer.

96. The method of claim 79, wherein the polymerase is a DNA polymerase or an RNA

polymerase.

97. The method of claim 79 or 96, wherein the polymerase is Taq polymerase.

98. The method of claim 97, wherein the Taq polymerase is native or modified Taq polymerase.

99. The method of any one of the claims 79-98, wherein the albumin binding moiety is

directly connected to the Taq polymerase or the albumin binding moiety is connected to the Taq polymerase though a spacer.

100. The method of any one of the claims 79-99, wherein a genetic sequence of the albumin binding moiety and the Taq polymerase comprises the albumin binding moiety sequence residing on the 3 ' end of the Taq polymerase sequence, residing on the 5 ' end of the Taq polymerase sequence, or residing on both the 3' end and 5' end of the Taq polymerase sequence.

101. The method of any one of the claims 79-100, wherein the Taq polymerase consists of a sequence selected from SEQ ID NO: 1.

102. The method of any one of the claims 79- 101, wherein the albumin binding moiety able to bind serum albumin is at least a part of Streptococcal protein G.

103. The method of claim 102, wherein the at least a part of Streptococcal protein G is the entire Streptococcal protein G.

104. The method of claim 102, wherein the at least a part of Streptococcal protein G

comprises ABP (121aa), BB (214aa), ABD (46aa), ADB1 binding site, ADB2 binding site, or ADB3 binding site.

105. The method of claim 104, wherein the ABD to albumin affinity is 1.5 nanomolar or less.

106. A method of removing a nucleic acid contaminant from a biological sample, comprising:

a) contacting a biological sample with protamine-coated beads; and

b) harvesting the biological sample from protamine-coated beads through a separation method to remove the nucleic acid contaminant from the biological sample.

107. The method of claim 106, wherein the biological sample is a protein sample.

108. The method of claim 106 or 107, wherein the biological sample is a polymerase sample.

109. The method of claim 108, wherein the polymerase sample is a DNA polymerase sample or an RNA polymerase sample.

110. The method of claim 108 or 109, wherein the polymerase sample is a Taq polymerase sample.

111. The method of claim 110, wherein the Taq polymerase is native or modified Taq

polymerase.

112. The method of claim 111, wherein the Taq polymerase consists of a sequence selected from SEQ ID NO: 1.

113. The method of claim 106, wherein the biological sample is a cell lysis sample.

114. The method of claim 106, wherein the biological sample is a culture media sample.

115. The method of claim 106, wherein the protamine-coated beads are beads covalently

bound to protamine.

116. The method of claim 106 or 115, wherein the beads are Sepharose beads or magnetic beads.

117. The method of claim 106, wherein the contacting comprises incubating the biological sample with protamine-coated beads for from about 2 min to about 24 hours.

118. The method of claim 106 or 117, wherein the contacting further comprises incubating the biological sample with protamine-coated beads at a buffer pH of from about 4 to about 9.

119. The method of any one of the claims 106, 117, or 118, wherein the contacting further comprises incubating about Ιμί to about ΙΟΟμί of protamine-coated beads with about 1 mg of biological sample.

120. The method of claim 106, wherein the separation method is a centrifugation method or column chromatography method.

121. The method of claim 106, wherein the nucleic acid contaminant is DNA contaminant.

122. The method of claim 106, wherein the method further comprises removing the nucleic acid contaminant through an electrophoretic method.

123. The method of claim 122, wherein the electrophoretic method is performed after

harvesting the biological sample from the protamine-coated beads.

124. The method of claim 106 or 122, wherein the method further comprises removing the nucleic acid contaminant through a silica-based method.

125. The method of claim 124, wherein the silica-based method comprises contacting a

growth media with silica and harvesting the silica-treated growth media with a separation method.

126. The method of any one of the claims 106-125, wherein protamine is obtained from

salmon.

127. An assay kit for determining the activity of a polymerase comprising an oligonucleotide selected from SEQ ID NOs: 8-10.

128. The assay kit of claim 127, wherein the polymerase is a DNA polymerase or an RNA polymerase.

129. The assay kit of claim 127 or 128, wherein the polymerase comprises Taq polymerase, Vent polymerase, Klenow Fragment (3 '-5' exo-), DNA Polymerase I (large Klenow fragment), E. coli DNA polymerase I, phi29 DNA polymerase, Phusion DNA

polymerase, or T4 DNA polymerase.

130. The assay kit of claim 129, wherein the polymerase is a Taq polymerase.

131. The assay kit of claim 130, wherein the Taq polymerase is native or modified Taq

polymerase.

132. The assay kit of any one of the claims 127-131, wherein the assay kit further comprises a primer.

133. The assay kit of claim 127, wherein the activity of the polymerase is determined from an amplification reaction.

134. The assay kit of claim 133, wherein a pyrophosphate is released during the amplification reaction.

135. The assay kit of claim 134, wherein the rate of pyrophosphate release during the

amplification reaction is used to determine the activity of the polymerase.