Multiplex amplification and separation of nucleic acid sequences using ligation-dependant strand displacement amplification an bioelectronic chip technology

• US 6,864,071 B2
• Filed: 05/25/2001
• Issued: 03/08/2005
• Est. Priority Date: 04/12/1999
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen/Becton Dickinson Partnership
• Inventors: Gerrue, Louis O., Diver, Jonathan M., Carrino, John J.
• Primary Examiner(s): Myers, Carla J.
• Assistant Examiner(s): Sheinberg, Monika
• Application Number:
  US09/865,807
• Publication Number:
  US 20020068334A1
• Time in Patent Office: 1,383 Days
• Field of Search: 435/6, 435/91.1, 435/912, 536/22.1, 536/23.1, 536/243, 536/25.32, 536/24.4, 536/27, 436/501, 436/94
• US Class Current: 435/91.2
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00614 Delimitation of the attachment areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to devices, methods, and compositions of matter for the multiplex amplification and analysis of nucleic acid sequences in a sample using ligation-dependent strand displacement amplification technologies in combination with bioelectronic microchip technology.

Claims

1. A method for the amplification of at least one target nucleic acid sequence of interest, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer, and no bumper primers are used in the strand displacement amplification,

wherein steps (a), (b), and (c) exit in solution at the same time.

2. A method for the amplification of at least one target nucleic acid sequence of interest, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact. with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first and a second SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer, and the strand displacement reaction utilizes an unequal concentration ratio of the first SDA primer to the second SDA primer, wherein the first and second SDA primers are different primers and form a set of primers, and wherein unequal populations of complementary first and second amplified strands of the target nucleic acids are formed,

wherein steps (a), (b), and (c) occur at the same time.

3. The method of claim 2 wherein the unequal concentration ratio is obtained by providing at least one SDA primer of the set in molar excess as compared to the other SDA primer in the set.

4. The method of claim 3 wherein the SDA primer that is in molar excess is labeled.

5. The method of claim 4 wherein the label is a fluorescent label.

6. The method of claim 5 wherein the fluorescent label is selected from the group consisting of Bodipy-derivatives, Cyanine-derivatives, fluorescein-derivatives, and rhodamine-derivatives.

7. The method of claim 4 wherein the label is a chemiluminescence label.

8. The method of claim 4 wherein the label is an electrochemiluminescence label.

9. The method of claim 4 wherein the label is biotin.

10. The method of claim 2 wherein the unequal concentration ratio is obtained by providing a competitor in the amplification reaction to either the first SDA primer or the second SDA primer, and wherein the competitor binds to the ligated target probe template and is selected from the group consisting of:

non-extendable competitors, non-cleavable competitors, and competitors which are non-extendable and non-cleavable.

11. The method of claim 10 wherein the competitor is anchored to a substrate.

12. The method of claim 10 wherein the competitor is in solution.

13. The method of claim 10 wherein the competitor is a non-extendable competitor.

14. The method of claim 13 wherein the competitor comprises a non-extendable 3′

modification selected from the group consisting of;

a 3′

terminal base mis-match, a 3′

dideoxy nucleic acid, and a blocking group attached to the 3′

hydroxy group of the 3′

terminal nucleic acid.

15. The method of claim 10 wherein the competitor is a non-cleavable competitor.

16. The method of claim 15 wherein the non-cleavable competitor comprises a nucleic acid selected from the group consisting of:

methylated nucleic acids and phosphorothiolated nucleic acid.

17. The method of claim 15 wherein the non-cleavable competitor comprises a modified nucleic acid selected from the group consisting of 2′

deoxyadenosine 5′

-O-(1-thiotriphosphate), 5-methyldeoxycytidine 5′

-triphosphate, 2′

-deoxyuridine 5′

-triphosphate, and 7-deaza-2′

deoxyguanosine 5′

-triphosphate.

18. The method of claim 10 wherein the SDA primer which the competitor does not compete with is labeled.

19. The method of claim 18 wherein the label is a fluorescent label.

20. The method of claim 19 wherein the fluorescent label is selected from the group consisting of Bodipy-derivatives, Cyanine-derivatives, fluorescein-derivatives, and rhodamine-derivatives.

21. The method of claim 18 wherein the label is a chemiluminescence label.

22. The method of claim 18 wherein the label is an electrochemiluminescence label.

23. The method of claim 18 wherein the label is biotin.

24. A method for the amplification of at least one target nucleic acid sequence of interest, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer and a second SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5'"'"' region of the primer, wherein the fist and second SDA primers are different primers,

wherein steps (a), (b), and (c) occur at the same time and at least one of the first and second SDA primers is labeled.

25. The method of claim 24 wherein the label is a fluorescent label.

26. The method of claim 25 wherein the fluorescent label is selected from the group consisting of Bodipy-derivatives, Cyanine-derivatives, fluorescein-derivatives, and rhodamine-derivatives.

27. The method of claim 24 wherein the label is a chemiluminescence label.

28. The method of claim 24 wherein the label is an electrochemiluminescence label.

29. The method of claim 24 wherein the label is biotin.

30. A method for the amplification of at least one target nucleic acid sequence of interest, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer,

wherein steps (a), (b), and (c) exist in solution at the same time,

wherein allele specific strand displacement amplification is carried out for target nucleic acids of interest which encode a gene with two or more known alleles,

wherein the upstream and downstream ligation probe sequences are selected to be specific for a particular allele of the gene, so that the ligation probes contact, proximate the 3′

end of the upstream probe or proximate the 5′

end of the downstream probe, a nucleotide sequence of the target nucleic acid sequence which is determinative of the particular allele,

wherein, if the target nucleic acid does not contain the sequence determinative of the allele for which the ligation probes are selected, the juxtaposed terminus of one of the probes is misaligned, so that, in step (b), the ligation probes are ligated only if the target nucleic acid sequence contains the allele determinative sequence, and

wherein, in step (c), amplicons are produced by strand displacement amplification if the allele determinative sequence is contained within the target nucleic acid sequence.

31. The method of claim 30 wherein the upstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the ultimate 3′

nucleic acid of the upstream probe.

32. The method of claim 30 wherein the upstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the penultimate 3′

nucleic acid of the upstream probe.

33. The method of claim 30 wherein the downstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the ultimate 5′

nucleic acid of the downstream probe.

34. The method of claim 30 wherein the downstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the penultimate 5′

nucleic acid of the downstream probe.

35. A method for the amplification of at least one target nucleic acid sequence of interest, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence, wherein the upstream and downstream oligonucleotide ligation probes are initially incapable of being ligated together;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, wherein each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer,

wherein steps (a), (b), and (c) exist in solution at the same time and the method further comprising rendering capable of being ligated together the upstream and downstream oligonucleotide ligation probes prior to ligating the probes together in step (b).

36. The method of claim 35 wherein the rendering comprises removing one or more terminal nucleotides from the oligonucleotide probes.

37. The method of claim 36 wherein one or more terminal nucleotides are removed from the 3′

terminus of the upstream oligonucleotide ligation probe.

38. The method of claim 37 wherein one or more terminal nucleotides are removed by an endonuclease.

39. The method of claim 38 wherein the endonuclease is Endonuclease IV.

40. The method of claim 36 wherein one or more terminal nucleotides are removed from the 5′

terminus of the downstream oligonucleotide ligation probe.

41. The method of claim 40 wherein one or more terminal nucleotides are removed by an exonuclease.

42. The method of claim 41 wherein the exonuclease is a DNA polymerase.

43. The method of claim 36 wherein one or more terminal nucleotides that are removed are non-complementary to the target sequence.

44. A method for the amplification of at least one target nucleic acid sequence of interest from at least one sample, using an electronically addressable microchip, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer, and no bumper primers are used in the strand displacement amplification,

wherein at least one nucleic acid selected from group consisting of target sequences, ligated target probe templates, and amplicons is electronically addressed in a step (d) to any of a plurality of capture sites on the bioelectronic microchip.

45. A method for the amplification of at least one target nucleic acid sequence of interest from at least one sample, using an electronically addressable microchip, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer and a second SDA primer, the first and second SDA primers being different primers, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer, and the first and second SDA primers are contained within a branched primer structure, having the capacity to support strand displacement amplification, and the branched primer is attached to a capture site,

wherein at least one nucleic acid selected from group consisting of target sequences, ligated target probe templates, and amplicons is electronically addressed in a step (d) to any of a plurality of capture sites on the bioelectronic microchip.

46. The method of claim 45 wherein the branched primer is attached to the capture site through a biotin/streptavidin interaction.

47. The method of claim 45 wherein the branched primer is attached to the capture site through a covalent bond.

48. The method of claim 45 wherein the branched primer is formed by attaching the first and second SDA primers to an amino acid.

49. The method of claim 48 wherein the amino acid is lysine.

50. The method of claim 45 wherein the branched primer formed by attaching the first and second SDA primers to a spacer molecule selected from the group consisting of polyethylene glycol polymers, polyamino acids, and a polyfunctional amino acid which has been derivatized with two nucleic acid oligomers.

51. A method for the amplification of at least one target nucleic acid sequence of interest from at least one sample, using an electronically addressable microchip, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer,

wherein at least one nucleic acid selected from group consisting of target sequences, ligated target probe templates, and amplicons is electronically addressed in a step (d) to any of a plurality of capture sites on the bioelectronic microchip and the upstream and downstream oligonucleotide ligation probes are initially incapable of being ligated together, the method further comprising rendering capable of being ligated together the upstream and downstream oligonucleotide ligation probes prior to ligating the probes together in step (b).

52. The method of claim 51 wherein the rendering comprises removing one or more terminal nucleotides from the oligonucleotide probes.

53. The method of claim 52 wherein one or more terminal nucleotides are removed from the 3′

terminus of the upstream oligonucleotide ligation probe.

54. The method of claim 53 wherein one or more terminal nucleotides are removed by an endonuclease.

55. The method of claim 54 wherein the endonuclease is Endonuclease IV.

56. The method of claim 52 wherein one or more terminal nucleotides are removed from the 5′

terminus of the downstream oligonucleotide ligation probe.

57. The method of claim 56 wherein one or more terminal nucleotides are removed by an exonuclease.

58. The method of claim 57 wherein the exonuclease is a DNA polymerase.

59. The method of claim 52 wherein one or more terminal nucleotides that are removed are non-complementary to the target sequence.

60. A method for the amplification of at least one target nucleic acid sequence of interest from at least one sample, using an electronically addressable microchip, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer and a second SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer, and the strand displacement amplification utilizes an unequal concentration ratio of the first SDA primer to the second SDA primer, wherein the first and second SDA primers are different primers and form a set of primers, and wherein unequal populations of complementary first and second amplified strands of the target nucleic acids are formed,

wherein at least one nucleic acid selected from group consisting of target sequences, ligated target probe templates, and amplicons is electronically addressed in a step (d) to any of a plurality of capture sites on the bioelectronic microchip.

61. The method of claim 60 wherein the unequal concentration ratio is obtained by providing at least one SDA primer of the set of primers in molar excess as compared to the other SDA primer in the set of primers.

62. The method of claim 61 wherein the SDA primer that is in molar excess is labeled.

63. The method of claim 62 wherein the label is a fluorescent label.

64. The method of claim 63 wherein the fluorescent label is selected from the group consisting of Bodipy-derivatives, Cyanine-derivatives, fluorescein-derivatives, and rhodamine-derivatives.

65. The method of claim 62 wherein the label is a chemiluminescence label.

66. The method of claim 62 wherein the label is an electrochemiluminescence label.

67. The method of claim 62 wherein the label is biotin.

68. The method of claim 60 wherein the unequal concentration ratio is obtained by providing a competitor in the amplification reaction to either the first SDA primer or the second SDA primer, and wherein the competitor binds to the ligated target probe template and is selected from the group consisting of:

non-extendable competitors, non-cleavable competitors, and competitors which are non-extendable and non-cleavable.

69. The method of claim 68 wherein the competitor is anchored to a substrate.

70. The method of claim 68 wherein the competitor is in solution.

71. The method of claim 68 wherein the competitor is a non-extendable competitor.

72. The method of claim 71 wherein the competitor comprises a non-extendable 3′

modification selected from the group consisting of;

a 3′

terminal base mis-match, a 3′

dideoxy nucleic acid, and a blocking group attached to the 3′

hydroxy group of the 3′

terminal nucleic acid.

73. The method of claim 68 wherein the competitor is a non-cleavable competitor.

74. The method of claim 73 wherein the non-cleavable competitor comprises a nucleic acid selected from the group consisting of:

methylated nucleic acids and phosphorothiolated nucleic acid.

75. The method of claim 73 wherein the non-cleavable competitor comprises a modified nucleic acid selected from the group consisting of 2′

deoxyadenosine 5′

-O-(1-thiotriphosphate), 5-methyldeoxycytidine 5′

-triphosphate, 2′

-deoxyuridine 5′

-triphosphate, and 7-deaza-2′

-deoxyguanosine 5′

-triphosphate.

76. The method of claim 68 wherein the SDA primer which the competitor does not compete with is labeled.

77. The method of claim 76 wherein the label is a fluorescent label.

78. The method of claim 77 wherein the fluorescent label is selected from the group consisting of Bodipy-derivatives, Cyanine-derivatives, fluorescein-derivatives, and rhodamine-derivatives.

79. The method of claim 76 wherein the label is a chemiluminescence label.

80. The method of claim 76 wherein the label is an electrochemiluminescence label.

81. The method of claim 76 wherein the label is biotin.

82. A method for the amplification of at least one target nucleic acid sequence of interest from at least one sample, using an electronically addressable microchip, comprising, for each target nucleic acid sequence of interest:

(a) contacting upstream and downstream oligonucleotide ligation probes with the target nucleic acid sequence such that the 3′

terminus of the upstream probe is juxtaposed to the 5′

terminus of the downstream probe while both the upstream and downstream probes are in contact with the target nucleic acid sequence;

(b) ligating together the upstream and downstream probes at their respective juxtaposed termini to form a ligated target probe template; and

(c) using the ligated target probe template in a strand displacement amplification (“

SDA”

) reaction to form amplicons of the ligated target probe template, wherein the strand displacement reaction utilizes at least a first SDA primer, each SDA primer comprising a restriction endonuclease sequence on the 5′

region of the primer,

wherein at least one nucleic acid selected from group consisting of target sequences, ligated target probe templates, and amplicons is electronically addressed in a step (d) to any of a plurality of capture sites on the bioelectronic microchip,

wherein allele specific strand displacement amplification is carried out for target nucleic acids of interest which encode a gene with two or more known alleles,

wherein the upstream and downstream ligation probe sequences are selected to be specific for a particular allele of the gene, so that the ligation probes contact, proximate the 3′

end of the upstream probe or proximate the 5′

end of the downstream probe, a nucleotide sequence of the target nucleic acid sequence which is determinative of the particular allele,

wherein, if the target nucleic acid does not contain the sequence determinative of the allele for which the ligation probes are selected, the juxtaposed terminus of one of the probes is misaligned, so that, in step (b), the ligation probes are ligated only if the target nucleic acid sequence contains the allele determinative sequence, and

wherein, in step (c), amplicons are produced by strand displacement amplification if the allele determinative sequence is contained within the target nucleic acid sequence.

83. The method of claim 82 wherein the gene has two known alleles, and the target nucleic acids are addressed to two capture sites.

84. The method of claim 82 wherein the gene has multiple known alleles, and the target nucleic acids are addressed to multiple capture sites.

85. The method of claim 82 wherein the upstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the ultimate 3′

nucleic acid of the upstream probe.

86. The method of claim 82 wherein the upstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the penultimate 3′

nucleic acid of the upstream probe.

87. The method of claim 82 wherein the downstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the ultimate 5′

nucleic acid of the downstream probe.

88. The method of claim 82 wherein the downstream probe is particular for the allele determinative sequence, and is not complementary for other alleles at the penultimate 5′

nucleic acid of the downstream probe.