Direct multiplex characterization of genomic DNA

• US 20070264642A1
• Filed: 06/30/2006
• Published: 11/15/2007
• Est. Priority Date: 10/24/2000
• Status: Abandoned Application

• Current Assignee: Maneesh Jain, Mostafa Ronaghi, Paul Hardenbol, Ronald Davis, Thomas Willis, Viktor Stolc
• Original Assignee: Maneesh Jain, Mostafa Ronaghi, Paul Hardenbol, Ronald Davis, Thomas Willis, Viktor Stolc
• Inventors: Jain, Maneesh, Ronaghi, Mostafa, Stolc, Viktor, Hardenbol, Paul, Willis, Thomas, Davis, Ronald
• Application Number:
  US11/480,270
• Publication Number:
  US 20070264642A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• 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

The invention is directed to novel methods of multiplexing nucleic acid reactions, including amplification, detection and genotyping. The invention relies on the use of precircle probes that are circularized in the presence of the corresponding target nucleic acids, cleaved, and then amplified.

Claims

1-47. -47. (canceled)

48. A composition comprising a plurality of padlock probes for detecting a plurality of target sequences in a sample, wherein each target sequence comprises first and second target domains, and each of the padlock probes comprises:

a) a first probe sequence complementary to the first target domain;

b) a second probe sequence complementary to the second target domain;

c) a priming site that is identical for each of the plurality of probes; and

d) a restriction endonuclease site that is identical for each of the plurality of probes, wherein the restriction site occurs in a sequence intervening between the first probe sequence and the second probe sequence.

49. The composition of claim 48, wherein each padlock probe also comprises an adapter sequence.

50. The composition of claim 49, wherein the adapter sequence is unique to a specific combination of first and second probe sequences in a padlock probe.

51. The composition of claim 49, wherein the adapter sequence occurs in a sequence intervening between the first probe sequence and the second probe sequence.

52. The composition of claim 48, wherein the padlock probe has a first terminus comprising the first probe sequence and a second terminus comprising the second probe sequence.

53. The composition of claim 52, wherein the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.

54. The composition of claim 53, wherein a circularized probe can be formed by ligation of the padlock probe.

55. The composition of claim 53, wherein a circularized probe can be formed by extension and ligation of the padlock probe.

56. The composition of claim 48, wherein the padlock probe is circularized.

57. The composition of claim 56, wherein the padlock probe has no terminus.

58. The composition of claim 48, wherein the first target domain comprises a single nucleotide polymorphism (SNP).

59. A composition comprising a plurality of padlock probes for detecting a plurality of target sequences in a sample, wherein each target sequence comprises first and second target domains, and each padlock probe comprises:

a) a first terminus comprising a first probe sequence complementary to the first target domain, wherein said first probe sequence is unique for each of the plurality of probes;

b) a second terminus comprising a second probe sequence complementary to the second target domain, wherein said second probe sequence is unique for each of the plurality of probes;

c) two priming sites that are identical for each of the plurality of probes; and

d) a restriction endonuclease site that is identical for each of the plurality of probes, wherein the restriction site occurs in a sequence intervening between the first probe sequence and the second probe sequence.

60. The composition of claim 59, wherein each padlock probe also comprises a barcode sequence.

61. The composition of claim 60, wherein the barcode sequence is unique to a specific combination of first and second probe sequences in a padlock probe.

62. The composition of claim 60, wherein the barcode sequence occurs in a sequence intervening between the first probe sequence and the second probe sequence.

63. The composition of claim 59, wherein the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.

64. The composition of claim 59, wherein a circularized probe can be formed by ligation of the first terminus and the second terminus of the padlock probe when the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.

65. The composition of claim 59, wherein a circularized probe can be formed by extension of at least one terminus and ligation of the padlock probe when the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.

66. The composition of claim 59, wherein the padlock probe is circularized.

67. The composition of claim 66, wherein the padlock probe has no terminus.

68. The composition of claim 59, wherein the first target domain comprises a single nucleotide polymorphism (SNP).

69. A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of:

a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises;

i) a first end that is complementary to said first domain of said target sequence;

ii) a barcode sequence;

iii) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe; and

iv) two priming sites

b) hybridizing said second domain of said target sequence to said second end of said padlock probe;

c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;

d) amplifying a sequence comprising at least a portion of said padlock probe following said covalently attaching, thereby producing amplification products comprising said barcode sequence; and

e) binding said barcode sequence of said amplification product to a substrate wherein said substrate comprises an attached barcode capture probe and/or an attached complement of said barcode sequence;

thereby identifying said nucleotide at said detection position.

70. A method according to claim 69, wherein said substrate is an array.

71. A method according to claim 70, wherein said array is prepared by spotting.

72. A method according to claim 70, wherein said array is prepared by photolithography.

73. A method according to claim 69, wherein said array is prepared from beads on a solid support.

74. The method according to claim 69 wherein said first end and said second end of said padlock probe are hybridized to said target sequence and said covalently attaching comprises ligating said first and second ends of said padlock probe.

75. The method according to claim 69 wherein one of said first end or said second end of said padlock probe hybridized to said target sequence is extended using a polymerase, and said covalently attaching comprises ligating said first and second ends of said padlock probe after said first or second end is extended.

76. The method according to claim 69 wherein said detection position is an SNP.

77. The method according to claim 69 wherein after said covalently attaching said first end of said padlock probe and said second end of said padlock probe, a sequence comprising at least a portion of said padlock probe is amplified with a polymerase, then cleaved.

78. The method according to claim 69 wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.

79. The method according to claim 69 wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.

93. The method according to claim 69 wherein said amplifying comprises polymerase chain reaction.

80. A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of:

a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises;

i) a first end that is complementary to said first domain of said target sequence;

ii) a barcode sequence;

iii) a cleavage site;

iv) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe; and

v) two priming sites;

b) hybridizing said second domain of said target sequence to said second end of said padlock probe;

c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;

d) cleaving said covalently attached padlock probe at said cleavage site; and

e) binding said barcode sequence of said covalently attached padlock probe to a substrate wherein said substrate comprises a capture probe and/or an attached complement of said adapter sequence;

thereby identifying said nucleotide at said detection position.

81. A method according to claim 80, wherein said substrate is an array.

82. A method according to claim 81, wherein said array is prepared by spotting.

83. A method according to claim 81, wherein said array is prepared by photolithography.

84. A method according to claim 81, wherein said array is prepared from beads on a solid support.

85. The method according to claim 80 wherein said first end and said second end of said padlock probe are hybridized to said target sequence and said covalently attaching comprises ligating said first and second ends of said padlock probe.

86. The method according to claim 80 wherein one of said first end or said second end of said padlock probe hybridized to said target sequence is first extended using a polymerase, and said covalently attaching comprises ligating said first and second ends of said padlock probe after said first or second end is extended.

87. The method according to claim 80 wherein said detection position is an SNP.

88. The method according to claim 80 further comprising amplifying a sequence comprising at least a portion of said padlock probe with a polymerase following said covalently attaching, thereby producing amplification products comprising said barcode sequence.

89. The method according to claim 80 wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.

90. The method according to claim 80 wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.

91. The method according to claim 88 further comprising binding said barcode sequence of said amplification products to said substrate that comprises an attached capture probe and/or an attached complement of said adapter sequence.

92. The method according to claim 88 wherein said amplifying comprises polymerase chain reaction.