Massive parallel method for decoding DNA and RNA

• US 20100317012A1
• Filed: 07/19/2010
• Published: 12/16/2010
• Est. Priority Date: 10/06/2000
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Li, Zengmin, Edwards, John Robert, Itagaki, Yasuhiro
• Granted Patent: US 8,088,575 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

Claims

1-60. -60. (canceled)

61. A nucleotide analogue which comprises:

(a) a base selected from the group consisting of adenine or an analogue of adenine, cytosine or an analogue of cytosine, guanine or an analogue of guanine, thymine or an analogue of thymine, and uracil or an analogue of uracil;

(b) a unique label attached through a cleavable linker to the base or to an analogue of the base;

(c) a deoxyribose; and

(d) a small cleavable chemical group to cap an —

OH group at a 3′

-position of the deoxyribose.

62. The nucleotide analogue of claim 61, wherein said small cleavable chemical group is less than 300 daltons in mass.

63. The nucleotide analogue of claim 61, wherein said small cleavable chemical group comprises two carbon atoms or three carbon atoms.

64. The nucleotide analogue of claim 63, wherein said cleavable chemical group is —

CH₂OCH₃ or —

CH₂CH═

CH₂.

65. The nucleotide analogue of claim 61, wherein said base is a deazapurine.

66. A nucleotide analogue which comprises:

(a) a base selected from the group consisting of adenine or an analogue of adenine, cytosine or an analogue of cytosine, guanine or an analogue of guanine, thymine or an analogue of thymine, and uracil or an analogue of uracil;

(b) a unique label attached through a cleavable linker to the base or to an analogue of the base;

(c) a deoxyribose; and

(d) a small cleavable chemical group to cap an —

OH group at a 3′

-position of the deoxyribose, wherein said cleavable chemical group does not interfere with the recognition of the nucleotide analogue by polymerase as a substrate.

67. The nucleotide analogue of claim 66, wherein said small cleavable chemical group is less than 300 daltons in mass.

68. The nucleotide analogue of claim 66, wherein said small cleavable chemical group comprises two carbon atoms or three carbon atoms.

69. The nucleotide analogue of claim 68, wherein said cleavable chemical group is —

CH₂OCH₃ or —

CH₂CH═

CH₂.

70. The nucleotide analogue of claim 66, wherein said base is a deazapurine.

71. A nucleotide analogue which comprises:

(a) a base selected from the group consisting of adenine or an analogue of adenine, cytosine or an analogue of cytosine, guanine or an analogue of guanine, thymine or an analogue of thymine, and uracil or an analogue of uracil;

(b) a unique label attached through a cleavable linker to the base or to an analogue of the base;

(c) a deoxyribose; and

(d) a cleavable chemical group to cap an —

OH group at a 3′

-position of the deoxyribose, said chemical group comprising three carbons or less.

72. The nucleotide analogue of claim 71, wherein said small cleavable chemical group comprises two carbon atoms or three carbon atoms.

73. The nucleotide analogue of claim 72, wherein said cleavable chemical group is —

CH₂OCH₃ or —

CH₂CH═

CH₂.

74. The nucleotide analogue of claim 71, wherein said base is a deazapurine.

75. A method of determining the identity of a nucleotide analogue incorporated into a nucleic acid primer extension strand, comprising:

a) contacting a nucleic acid template attached to a solid surface with a nucleic acid primer which hybridizes to the template;

b) simultaneously contacting the product of step a) with a polymerase and four nucleotide analogues which are either (i) aA, aC, aG, and aT, or (ii) aA, aC, aG, and aU, so as to incorporate one of the nucleotide analogues onto the nucleic acid primer and form a nucleic acid primer extension strand, wherein each nucleotide analogue within (i) or (ii) comprises a base labeled with a unique label and contains a small removable chemical moiety capping the 3′

-OH group of the sugar of the nucleotide analogue, wherein said small cleavable chemical group does not interfere with the recognition of the nucleotide analogue by polymerase as a substrate; and

c) detecting the unique label of the incorporated nucleotide analogue, so as to thereby determine the identity of the nucleotide analogue incorporated into the nucleic acid primer extension strand.

76. The method of claim 75, further comprising removing the chemical moiety capping the 3′

-OH group of the sugar of the incorporated nucleotide analogue, thereby permitting the incorporation of a further nucleotide analogue so as to create a growing annealed nucleic acid primer extension strand.

77. The method of claim 75, wherein each of the four unique labels is a fluorescent label.

78. The method of claim 77, wherein one of said four unique labels comprises ROX.

79. The method of claim 77, wherein one of said four unique labels comprises R6G.

80. The method of claim 75, wherein said base of at least one of said nucleotide analogues is a deazapurine.