Method for the detection of multiple genetic targets

• US 20040110138A1
• Filed: 12/10/2002
• Published: 06/10/2004
• Est. Priority Date: 11/01/2002
• Status: Abandoned Application

• Current Assignee: University of Ottawa
• Original Assignee: University of Ottawa
• Inventors: Lem, Paul, Spiegelman, Jamie
• Application Number:
  US10/315,217
• Publication Number:
  US 20040110138A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• 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

Abstract

A method for simultaneous amplification and detection of multiple genetic targets is provided. Furthermore, a primer design protocol specific to the PCR reaction conditions of the present invention is also provided. The method of the present invention includes a PCR reaction mixture and primers specifically selected according to the reactions conditions provided. Multiple genetic targets are amplified simultaneously by this method, without requiring optimization of the reaction conditions.

Claims

1. A method for simultaneously amplifying multiple genetic targets, said method comprising:

selecting primer pairs specific to said multiple genetic targets according to a primer selection criteria;

effecting a hot start initiation of amplification of said multiple genetic targets in a single reaction vessel together with said primer pairs and a PCR reaction mixture;

performing a series of PCR reaction steps to amplify each of said multiple genetic sequences in said sample;

wherein said PCR reaction mixture is adaptable for simultaneously amplifying multiple genetic targets in said single reaction vessel without requiring optimization of pre-set amplification reaction conditions of said method when said primer pairs are provided at a final concentration of approximately 0.005 μ

M-0.05 μ

M in the reaction mixture.

2. The method of claim 1 wherein each of said primer pairs are provided to have a final concentration of approximately 0.01 μ

M in said reaction mixture.

3. The method of claim 1 wherein said PCR reaction mixture includes at least 5.0 mM MgCl₂.

4. The method of claim 1 wherein said primer selection criteria includes selecting primer pairs having a melting temperature in the range of 55 to 65°

C.

5. The method of claim 4 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

6. The method of claim 5 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

7. The method of claim 1 wherein said primer selection criteria includes selecting primer pairs having a GC content of 40-50%.

8. The method of claim 1 wherein said primer selection criteria includes selecting primer pairs that are 18-27 nucleotides in length.

9. The method of claim 1 wherein said series of PCR reaction steps includes a first and second PCR step.

10. The method of claim 1 wherein said series of PCR reaction steps includes a step of touchdown PCR.

11. The method of claim 10 wherein said step of touchdown PCR is performed in a first set of PCR steps.

12. The method claim 10 wherein said first set of PCR steps includes 20 cycles of touchdown PCR.

13. The method of claim 9 wherein said second set of reaction steps includes at least 20 cycles of PCR.

14. The method of claim 13 wherein said second set of reaction steps includes 25 cycles of PCR.

53. The method of claim 1 wherein said multiple genetic targets are DNA sequences.

54. The method of claim 53 wherein DNA sequences are selected from the group consisting of bacterial DNA, viral DNA, plant DNA, animal DNA or human DNA.

55. The method of claim 9 wherein said first series of PCR reaction steps comprises 20 cycles of touchdown PCR including 20 s at 95°

C., 1 min at 63°

C.—

decreased by 0.5 each cycle, and 1 min at 72°

C.

56. The method of claim 9 wherein said second series of PCR reaction steps comprises 25 cycles of PCR, including 20 s at 95°

C., 45 s at 56°

C. and 1 min at 72°

C.

57. The method of claim 56 including 7 min at 72°

C.

58. The method of claim 9 wherein said multiple genetic targets are DNA sequences.

59. The method of claim 58 wherein DNA sequences are selected from the group consisting of bacterial DNA, viral DNA, plant DNA, animal DNA or human DNA.

15. A method for preparing a PCR reaction mixture for simultaneously multiplexing multiple genetic targets, said method comprising:

adjusting the final MgCl₂ concentration of a PCR buffer suitable for a PCR reaction to at least 5.0 mM; and

adding a hot start initiation means to said buffer;

wherein said PCR reaction mixture is adaptable for simultaneously multiplexing multiple genetic targets in the presence of pre-selected primer pairs having a final concentration of 0.005-0.05 μ

M when added to said mixture.

16. The method of claim 15 wherein each of said pre-selected primer pairs are provided to have a final concentration of approximately 0.01 μ

M in said reaction mixture.

17. The method of claim 15 wherein the final concentration of MgCl₂ in said PCR reaction mixture is approximately 7.5 mM.

18. The method of claim 15 wherein said pre-selected primer pairs are selected to be and 18-27 nucleotides in length;

have a melting temperature in the range of 55 to 65°

C.; and

a GC content of 40-50%.

19. The method of claim 18 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

20. The method of claim 19 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

21. A method for simultaneously amplifying multiple genetic targets, said method comprising:

mixing a sample to be tested for the presence of said multiple genetic targets with pre-selected primer pairs specific to said multiple genetic targets and a PCR reaction mixture;

effecting a hot start initiation of amplification of said genetic targets;

performing a series of PCR reaction steps to amplify each of said multiple genetic sequences in said sample;

wherein said pre-selected primer pairs are provided to optimize amplification of said multiple genetic targets in said reaction mixture in the absence of requiring optimization of reaction conditions of said method.

22. The method of claim 21 wherein each of said primer pairs are provided to have a final concentration in said reaction mixture of 0.005-0.05 μ

M.

23. The method of claim 22 wherein each of said primer pairs are provided to have a final concentration of approximately 0.01 μ

M in said reaction mixture.

24. The method of claim 21 wherein said PCR reaction mixture includes at least 5.0 mM MgCl₂.

25. The method of claim 21 wherein said PCR reaction mixture includes approximately 7.5 mM MgCl₂.

26. The method of claim 21 wherein said pre-selected primer pairs are selected to be 18-27 nucleotides in length;

have a melting temperature in the range of 55 to 65°

C.; and

have a GC content of 40-50%.

27. The method of claim 26 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

28. The method of claim 27 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

29. The method of claim 21 wherein said series of PCR reaction steps includes a first and second PCR step.

30. The method of claim 21 wherein said series of PCR reaction steps includes a step of touchdown PCR;

31. The method of claim 30 wherein said step of touchdown PCR is performed in a first set of PCR steps.

32. The method claim 29 wherein said first set of PCR steps includes 20 cycles of touchdown PCR.

33. The method of claim 29 wherein said second set of reaction steps includes at least 20 cycles of PCR.

34. The method of claim 33 wherein said second set of reaction steps includes 25 cycles of PCR.

60. The method of claim 29 wherein said first series of PCR reaction steps comprises 20 cycles of touchdown PCR including 20 s at 95°

C., 1 min at 63°

C.—

decreased by 0.5 each cycle, and 1 min at 72°

C.

61. The method of claim 29 wherein said second series of PCR reaction steps comprises 25 cycles of PCR, including 20 s at 95°

C., 45 s at 56°

C. and 1 min at 72°

C.

62. The method of claim 61 including 7 min at 72°

C.

63. The method of claim 29 wherein said multiple genetic targets are DNA sequences.

64. The method of claim 63 wherein DNA sequences are selected from the group consisting of bacterial DNA, viral DNA, plant DNA, animal DNA or human DNA.

35. A method for simultaneously detecting multiple genetic targets, said method comprising:

mixing a sample to be tested for the presence of said multiple genetic targets with pre-selected primer pairs specific to said multiple genetic targets and a PCR reaction mixture;

effecting a hot start initiation of amplification of said genetic targets;

performing a series of PCR reaction steps to amplify each of said multiple genetic sequences in said sample; and

detecting said multiple genetic targets present in said sample;

wherein said pre-selected primer pairs are provided to optimize amplification of said multiple genetic targets in said PCR reaction mixture in the absence of requiring optimization of reaction conditions of said method.

36. The method of claim 35 wherein gel electrophoresis is employed in detecting said multiple genetic targets.

37. The method of claim 35 wherein each of said primer pairs are provided to have a final concentration in said reaction mixture of 0.005-0.05 M.

38. The method of claim 35 wherein each of said primer pairs are provided to have a final concentration of approximately 0.01 μ

M in said reaction mixture.

39. The method of claim 35 wherein said PCR reaction mixture includes at least 5.0 mM MgCl₂.

40. The method of claim 35 wherein said PCR reaction mixture includes approximately 7.5 mM MgCl₂.

41. The method of claim 35 wherein said pre-selected primer pairs are selected to be 18-27 nucleotides in length;

have a melting temperature in the range of 55 to 65°

C.; and

have a GC content of 40-50%.

42. The method of claim 41 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

43. The method of claim 42 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

44. The method of claim 35 wherein said series of PCR reaction steps includes a first and second PCR step.

45. The method of claim 35 wherein said serials of PCR reaction steps includes a step of touchdown PCR;

46. The method of claim 45 wherein said step of touchdown PCR is performed in a first set of PCR steps.

47. The method claim 44 wherein said first set of PCR steps includes 20 cycles of touchdown PCR.

48. The method of claim 44 wherein said second set of reaction steps includes at least 20 cycles of PCR.

49. The method of claim 43 wherein said second set of reaction steps includes 25 cycles of PCR.

50. The method of claim 44 wherein said first series of PCR reaction steps comprises 20 cycles of touchdown PCR including 20 s at 95°

C., 1 min at 63°

C.—

decreased by 0.5 each cycle, and 1 min at 72°

C.

51. The method of claim 44 wherein said second series of PCR reaction steps comprises 25 cycles of PCR, including 20 s at 95°

C., 45 s at 56°

C. and 1 min at 72°

C.

52. The method of claim 51 including 7 min at 72°

C.

65. A PCR reaction mixture for use in simultaneously amplifying multiple genetic targets, said mixture comprising:

a PCR buffer reagent including 5 mM-10 mM MgCl₂;

wherein said PCR reaction mixture is adaptable for simultaneously amplifying multiple genetic targets in a single reaction vessel without requiring optimization of pre-set amplification reaction conditions.

66. A PCR reaction mixture for use in simultaneously amplifying multiple genetic targets, said mixture comprising:

a PCR buffer reagent including 5 mM-10 mM MgCl₂;

wherein said PCR reaction mixture is adaptable for simultaneously amplifying multiple genetic targets in a single reaction vessel without requiring optimization of pre-set amplification reaction conditions.

67. A PCR reaction mixture for use in simultaneously amplifying multiple genetic targets in an amplification reaction, said mixture comprising:

a PCR buffer reagent including 5 mM-10 mM MgCl₂;

dNTPs having a final concentration of approximately 0.25 mM each; and

pre-selected primer pairs corresponding to target genetic sequences to be amplified;

each of said primers having a final concentration of approximately 0.005 μ

M-0.05 82 M in the reaction mixture;

wherein said PCR reaction mixture is adaptable for simultaneously amplifying multiple genetic targets in a single reaction vessel without requiring optimization of pre-set amplification reaction conditions.

68. A kit for simultaneously amplifying multiple genetic targets for detection, said kit comprising:

a PCR reaction mixture having a final MgCl₂ concentration of 5-12.5 mM;

a set of pre-selected, target-specific primer pairs corresponding to each of said multiple genetic targets; and

a set of instructions for using contents of said kit for simultaneously amplifying multiple genetic targets in a sample to be tested;

wherein said PCR reaction mixture is adaptable for simultaneously amplifying multiple genetic targets in a single reaction vessel without requiring optimization of pre-set amplification reaction conditions when said pre-selected, target-specific primer pairs are provided at a final concentration of approximately 0.005 μ

M -0.05 μ

M in the reaction mixture.

69. The kit of claim 68 wherein said PCR reaction mixture is pre-loaded in at least one reaction vessel.

70. The kit of claim 69 wherein said at least one reaction vessel further includes said set of pre-selected, target-specific primer pairs.

71. The kit of claim 68 further comprising, a DNA polymerase enzyme.

72. The kit of claim 71 wherein said DNA polymerase enzyme is a hot start enzyme.

73. A method for simultaneously amplifying multiple genetic targets, said method comprising:

mixing a sample to be tested for the presence of said multiple genetic targets with pre-selected primer pairs specific to said multiple genetic targets and a PCR reaction mixture including a final MgCl₂ concentration of at least 5.0 mM;

effecting means for a hot start initiation of amplification of said genetic targets; and

performing a series of PCR reaction steps including a step of touchdown PCR;

wherein said amplification is optimized when said pre-selected primer pairs are provided to have a final concentration of 0.005-005 μ

M in said reaction mixture.

74. The method of claim 73 wherein each of said pre-selected primer pairs are provided to have a final concentration of approximately 0.01 μ

M.

75. The method of claim 73 wherein said pre-selected primer pairs are selected to have a melting temperature in the range of 55 to 65°

C.

76. The method of claim 75 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

77. The method of claim 76 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

78. The method of claim 73 wherein each of said pre-selected primer pairs include a GC content of 40-50%.

79. The method of claim 73 wherein each of said pre-selected primer pairs is 18-27 nucleotides in length.

80. The method of claim 79 wherein each of said pre-selected primer pairs is 22 nucleotides in length.

81. The method of claim 73 wherein the final concentration of MgCl₂ in said PCR reaction mixture is 5 to 12.5 mM.

82. The method of claim 81 wherein said PCR reaction mixture includes more than 6 mM of MgCl₂.

83. The method of claim 82 wherein said PCR reaction mixture includes 7.5 mM of MgCl₂.

84. The method of claim 73 wherein ten or more genetic targets are simultaneously detected.

85. The method of claim 73 wherein said means for effecting a hot start initiation is a hot start enzyme.

86. The method of claim 85 wherein said hot start enzyme is a Taq Polymerase enzyme.

87. The method of claim 86 wherein said enzyme is Amplitaq Gold™

.

88. The method of claim 73 wherein said means for effecting a hot start initiation includes a DNA polymerase enzyme and a heating step.

111. The method of claim 73 wherein said series of PCR reaction steps includes 20 cycles of touchdown PCR including 20 s at 95°

C., 1 min at 63°

C.—

decreased by 0.5 each cycle, and 1 min at 72°

C.

112. The method of claim 111 wherein said series of PCR reaction steps further includes 25 cycles of PCR, including 20 s at 95°

C., 45 s at 56°

C. and 1 min at 72°

C.

113. The method of claim 112 further including 7 min at 72°

C.

114. The method of claim 73 wherein said genetic targets are DNA sequences.

115. The method of claim 114 wherein DNA sequences are selected from the group consisting of bacterial DNA, viral DNA, plant DNA, animal DNA or human DNA.

89. A method for simultaneously detecting multiple genetic targets in a sample to be tested, said method comprising:

selecting primer pairs corresponding to said multiple genetic targets according to a pre-defined primer selection criterion;

mixing said primer pairs with said sample to be tested and a PCR reaction mixtures;

said PCR reaction mixture including a final concentration of at least 5.0 mM MgCl₂;

effecting means for a hot start initiation of amplification of said genetic targets;

performing a series of PCR reaction steps including a step of touchdown PCR; and

detecting for the presence of said multiple genetic targets in said sample;

wherein when said primer pairs are provided in said reaction mixture to have a final concentration of 0.005-0.05 μ

M amplification of said multiple genetic targets is optimized in the absence of requiring optimization of reaction conditions of said method.

90. The method of claim 89 wherein each of said pre-selected primer pairs are provided to have a final concentration of approximately 0.01 μ

M.

91. The method of claim 89 wherein said pre-selected primer pairs are selected to have a melting temperature in the range of 55 to 65°

C.

92. The method of claim 91 wherein said melting temperatures of said pre-selected primer pairs are within a 2°

C. range of variation.

93. The method of claim 92 wherein the melting temperatures of said pre-selected primer pairs are 55-57°

C.

94. The method of claim 89 wherein each of said pre-selected primer pairs include a GC content of 40-50%.

95. The method of claim 89 wherein each of said pre-selected primer pairs is 18-27 nucleotides in length.

96. The method of claim 95 wherein each of said pre-selected primer pairs is 22 nucleotides in length.

97. The method of claim 89 wherein said PCR reaction mixture includes 5 to 12.5 mM of MgCl₂.

98. The method of claim 97 wherein said PCR reaction mixture includes 5 to 10 mM of MgCl₂.

99. The method of claim 98 wherein said PCR reaction mixture includes more than 6 mM of MgCl₂.

100. The method of claim 99 wherein said PCR reaction mixture includes 7.5 mM of MgCl₂.

101. The method of claim 89 wherein ten or more genetic targets are simultaneously detected.

102. The method of claim 89 wherein said means for effecting a hot start initiation is a hot start enzyme.

103. The method of claim 102 wherein said hot start enzyme is a Taq Polymerase enzyme.

104. The method of claim 103 wherein said enzyme is Amplitaq Gold™

.

105. The method of claim 89 wherein said means for effecting a hot start initiation includes a DNA polymerase enzyme and a heating step.

106. The method of claim 89 wherein said series of PCR reaction steps includes 20 cycles of touchdown PCR including 20 s at 95°

C., 1 min at 63°

C.—

decreased by 0.5 each cycle, and 1 min at 72°

C.

107. The method of claim 106 wherein said series of PCR reaction steps further includes 25 cycles of PCR, including 20 s at 95°

C., 45 s at 56°

C. and 1 min at 72°

C.

108. The method of claim 107 further including 7 min at 72°

C.

109. The method of claim 89 wherein said genetic targets are DNA sequences.

110. The method of claim 109 wherein DNA sequences are selected from the group consisting of bacterial DNA, viral DNA, plant DNA, animal DNA or human DNA.