Self-assembled single molecule arrays and uses thereof

• US 20100081128A1
• Filed: 09/29/2006
• Published: 04/01/2010
• Est. Priority Date: 10/07/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje, Yeung, George, Callow, Matthew J., Hauser, Brian K.
• Granted Patent: US 7,960,104 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides

Abstract

The present invention provides methods of making and using self-assembled arrays of single polynucleotide molecules for carrying out a variety of large-scale genetic measurements, such as gene expression analysis, gene copy number assessment, and the like. Random arrays used in the invention are “self-assembled” in the sense that they are formed by deposition of polynucleotide molecules onto a surface where they become fixed at random locations. The polynucleotide molecules fixed on the surface are then identified by direct sequence determination of component nucleic acids, such as incorporated probe sequences, or by other decoding schemes. Such identification converts a random array of determinable polynucleotides, and their respective probes into an addressable array of probe sequences.

Claims

1-58. -58. (canceled)

59. A method of identifying a sequence of a target polynucleotide, said method comprising:

(a) providing a substrate comprising a plurality of locations, wherein said plurality of locations each comprises a concatemer, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of said target polynucleotide;

(ii) a second target sequence of said target polynucleotide, wherein said first target sequence and said second target sequence are contiguous segments of said target polynucleotide;

(iii) a first interspersed adaptor interposed between said first target sequence and said second target sequence, wherein said first interspersed adaptor comprises a first anchor probe hybridization site adjacent to said first target sequence and a second anchor probe hybridization site adjacent to said second target sequence,

and wherein each concatemer comprises a different target polynucleotide;

(b) identifying a nucleotide of said first target sequence; and

(c) identifying a nucleotide of said second target sequence,

thereby identifying said sequence of said target polynucleotide.

61. A method according to claim 59, wherein said first interspersed adaptor comprises a palindromic sequence.

62. A method according to claim 59, wherein said first interspersed adaptor comprises a type IIs restriction endonuclease recognition site.

63. A method according to claim 59, wherein said identifying step (b) comprises:

(i) hybridizing a first anchor probe to said first anchor probe hybridization site;

(ii) hybridizing a first sequencing probe to said first t get sequence adjacent to said hybridized first anchor probe, wherein said first sequencing probe comprises a first detectable label;

(iii) ligating said hybridized first sequencing probe and said hybridized first anchor probe to form first ligated probes;

(iv) detecting said first ligated probes to identify said nucleotide of said first target sequence.

64. A method according to claim 63, wherein said first detectable label comprises a fluorophore.

65. A method according to claim 63, wherein said first sequencing probe is one of a set of probes comprising at least one degenerated position.

66. A method according to claim 63, wherein said steps (i) through (iv) are repeated multiple times to identify multiple nucleotides of said first target sequence.

67. A method according to claim 59, wherein said identifying step (c) comprises:

(i) hybridizing a second anchor probe to said second anchor probe hybridization site;

(ii) hybridizing a second sequencing probe to said second target sequence adjacent to said hybridized second anchor probe, wherein said second sequencing probe comprises a second detectable label;

(iii) ligating said hybridized second sequencing probe and said hybridized second anchor probe to form second ligated probes;

(iv) detecting said second ligated probes to identify said nucleotide of said second target sequence.

68. A method according to claim 67, wherein said second detectable label comprises a fluorophore.

69. A method according to claim 67, wherein said steps (i) through (iv) are repeated multiple times to identify multiple nucleotides of aid second target sequence.

70. A method according to claim 59,

wherein said identifying step (b) comprises:

(i) hybridizing a first anchor probe to said first anchor probe hybridization site;

(ii) hybridizing a first sequencing probe to said first target sequence adjacent to said hybridized first anchor probe, wherein said first sequencing probe comprises a first detectable label;

(iii) ligating said hybridized first sequencing probe and said hybridized first anchor probe to form first ligated probes;

(iv) detecting said first ligated probes to identify said nucleotide of said first target sequence,

and wherein said identifying step (c) comprises;

(i) hybridizing a second anchor probe to said second anchor probe hybridization site;

(ii) hybridizing a second sequencing probe to said second target sequence adjacent to said hybridized second anchor probe, wherein said second sequencing probe comprises a second detectable label;

(iii) ligating said hybridized second sequencing probe and said hybridized second anchor probe to form second ligated probes; and

(iv) detecting said second ligated probes to identify said nucleotide of said second target sequence.

71. A method according to claim 59, wherein said identifying step (b) and said identifying step (c) are performed simultaneously or sequentially.

72. A method according to claim 59, wherein each monomeric unit further comprises:

(a) a third target sequence of said target polynucleotide;

(b) a second interspersed adaptor interposed between said second target sequence and said third target sequence, wherein said second interspersed adaptor comprises a third anchor probe hybridization site and a fourth anchor probe hybridization site.

73. A method according to claim 72, wherein said method further comprises identifying a second nucleotide of said second target sequence and identifying a nucleotide of said third target sequence.

74. A method according to claim 73, wherein said identifying a second nucleotide of said second target sequence and said identifying a nucleotide of said third target sequence comprises:

(i) hybridizing a third anchor probe to said third anchor probe hybridization site;

(ii) hybridizing a third sequencing probe to said second target sequence adjacent to said hybridized third anchor probe, wherein said third sequencing probe comprises a third detectable label;

(iii) ligating said hybridized third sequencing probe and said hybridized third anchor probe to form third ligated probes;

(iv) detecting said third ligated probes to identify said second nucleotide of said second target sequence;

(v) hybridizing a fourth anchor probe to said fourth anchor probe hybridization site;

(vi) hybridizing a fourth sequencing probe to said third target sequence adjacent to said hybridized fourth anchor probe, wherein said fourth sequencing probe comprises a fourth detectable label;

(vii) ligating said hybridized fourth sequencing probe and said hybridized fourth anchor probe to form fourth ligated probes;

(viii) detecting said fourth ligated probes to identify said nucleotide of said third target sequence.

75. A method according to claim 72, wherein said second interspersed adaptor comprises a palindromic sequence.

76. A method according to claim 72, wherein said second interspersed adaptor comprises a type IIs endonuclease recognition site.

77. A method according to claim 59, wherein said target polynucleotide is all or part of a genome.

78. A method according to claim 59, wherein said substrate is formed by a method comprising:

providing a support comprising a surface, wherein said surface comprises said plurality of locations; and

immobilizing said concatemers on said surface such that said plurality of locations comprises a concatemer.

79. The method of claim 78, wherein said plurality of locations comprise reactive functionalities and said concatemers comprise functionalities complementary to said reactive functionalities, and wherein said immobilizing step occurs by covalent linkages formed between said reactive functionalities and said complementary functionalities.

80. The method of claim 78, wherein said immobilizing step comprises a noncovalent interaction between said concatemers and said plurality of locations.

81. The method of claim 80, wherein said noncovalent interaction is a member selected from:

Van der Waal force, hydrogen bonding, ionic interaction, and hydrophobic interaction.

82. The method of claim 78, wherein said concatemers are formed by a method comprising:

(a) providing fragments of said target polynucleotide;

(b) ligating an adaptor to a terminus of a plurality of said fragments;

(c) circularizing said fragments ligated to said adaptors to form circular products;

(d) generating a concatemer from at least one of said circular products, thereby forming said concatemers.

83. The method of claim 82, wherein said target polynucleotide is genomic DNA.

84. The method of claim 82, wherein said adaptor comprises a palindromic sequence.

85. The method of claim 82, wherein said adaptor comprises a type IIs endonuclease recognition site.

86. The method of claim 82, wherein said fragments substantially cover said target polynucleotide.

87. The method of claim 82, wherein said generating step (d) is accomplished using rolling circle replication.

60. (canceled)

88. A method of identifying a sequence of a target polynucleotide, said method comprising:

(a) providing a substrate comprising a plurality of locations, wherein said plurality of locations each comprises a concatemer, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of said target polynucleotide;

(ii) a second target sequence of said target polynucleotide, wherein said first target sequence and said second target sequence are contiguous segments of said target polynucleotide;

(iii) a first interspersed adaptor interposed between said first target sequence and said second target sequence, wherein said first interspersed adaptor comprises a first anchor probe hybridization site adjacent to said first target sequence and a second anchor probe hybridization site adjacent to said second target sequence,

and wherein each concatemer comprises a different target polynucleotide;

(b) identifying a nucleotide of said first target sequence by a method comprising;

(i) hybridizing a first anchor probe to said first anchor probe hybridization site;

(ii) hybridizing a first sequencing probe to said first target sequence adjacent to said hybridized first anchor probe, wherein said first sequencing probe comprises a first detectable label;

(iii) ligating said hybridized first sequencing probe and said hybridized first anchor probe to form first ligated probes;

(iv) detecting said first ligated probes to identify said nucleotide of said first target sequence; and

(c) identifying a nucleotide of said second target sequence by a method comprising;

(i) hybridizing a second anchor probe to said second anchor probe hybridization site;

(ii) hybridizing a second sequencing probe to said second target sequence adjacent to said hybridized second anchor probe, wherein said second sequencing probe comprises a second detectable label;

(iii) ligating said hybridized second sequencing probe and said hybridized second anchor probe to form second ligated probes; and

(iv) detecting said second ligated probes to identify said nucleotide of said second target sequence,

thereby identifying said sequence of said target polynucleotide.

89. A method of identifying a sequence of a target polynucleotide, said method comprising:

(a) providing a substrate comprising a plurality of locations, wherein said plurality of locations each comprises a concatemer, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of said target polynucleotide;

(ii) a second target sequence of said target polynucleotide, wherein said first target sequence and said second target sequence are contiguous segments of said target polynucleotide;

(iii) a first interspersed adaptor interposed between said first target sequence and said second target sequence, wherein said first interspersed adaptor comprises a first anchor probe hybridization site adjacent to said first target sequence and a second anchor probe hybridization site adjacent to said second target sequence;

(iv) a third target sequence of said target polynucleotide;

(v) a second interspersed adaptor interposed between said second target sequence and said third target sequence, wherein said second interspersed adaptor comprises a third anchor probe hybridization site and a fourth anchor probe hybridization site;

and wherein each concatemer comprises a different target polynucleotide;

(b) identifying a nucleotide of said first target sequence by a method comprising;

(i) hybridizing a first anchor probe to said first anchor probe hybridization site;

(ii) hybridizing a first sequencing probe to said first target sequence adjacent to said hybridized first anchor probe, wherein said first sequencing probe comprises a first detectable label;

(iii) ligating said hybridized first sequencing probe and said hybridized first anchor probe to form first ligated probes;

(iv) detecting said first ligated probes to identify said nucleotide of said first target sequence; and

(c) identifying a nucleotide of said second target sequence by a method comprising;

(i) hybridizing a second anchor probe to said second anchor probe hybridization site;

(ii) hybridizing a second sequencing probe to said second target sequence adjacent to said hybridized second anchor probe, wherein said second sequencing probe comprises a second detectable label;

(iii) ligating said hybridized second sequencing probe and said hybridized second anchor probe to form second ligated probes; and

(iv) detecting said second ligated probes to identify said nucleotide of said second target sequence,

(d) identifying a second nucleotide of said second target sequence and identifying a nucleotide of said third target sequence by a method comprising;

(i) hybridizing a third anchor probe to said third anchor probe hybridization site;

(ii) hybridizing a third sequencing probe to said second target sequence adjacent to said hybridized third anchor probe, wherein said third sequencing probe comprises a third detectable label;

(iii) ligating said hybridized third sequencing probe and said hybridized third anchor probe to form third ligated probes;

(iv) detecting said third ligated probes to identify said second nucleotide of said second target sequence;

(v) hybridizing a fourth anchor probe to said fourth anchor probe hybridization site;

(vi) hybridizing a fourth sequencing probe to said third target sequence adjacent to said hybridized fourth anchor probe, wherein said fourth sequencing probe comprises a fourth detectable label;

(vii) ligating said hybridized fourth sequencing probe and said hybridized fourth anchor probe to form fourth ligated probes;

(viii) detecting said fourth ligated probes to identify said nucleotide of said third target sequence;

thereby identifying said sequence of said target polynucleotide.