MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA

• US 20150197800A1
• Filed: 03/27/2015
• Published: 07/16/2015
• 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, Itagaki, Yasuhiro, Edwards, John Robert
• Granted Patent: US 10,669,577 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

1-48. -48. (canceled)

49. A method for simultaneously sequencing a plurality of different deoxyribonucleic acids, wherein the plurality of different deoxyribonucleic acids is covalently immobilized on a solid surface, and wherein a sequencing method by synthesis comprising a plurality of cycles, each cycle having a plurality of steps, is simultaneously applied to each of said covalently immobilized different deoxyribonucleic acids, said sequencing method involving the detection of the identity of a plurality of nucleotide analogues incorporated into a plurality of growing strands of DNA hybridized to deoxyribonucleic acids, said method comprising:

(a) providing to the plurality of different deoxyribonucleic acids more than one type of nucleotide analogue selected from the group consisting of aA, aC, aG, aT, and aU, wherein each type of nucleotide analogue is labeled with a unique label attached through a cleavable linker to the base and contains a chemical moiety capping the 3′

-OH group, wherein said chemical moiety is removable by chemical means,

under conditions such that a plurality of growing strands are extended by incorporation of one nucleotide analogue per strand so as to create a plurality of extended growing strands of DNA using a DNA polymerase reaction, said incorporated analogues serving as terminators of the polymerase reaction;

(b) detecting said unique label of said incorporated nucleotide analogues, so as to thereby identify 10,000 or more of the nucleotide analogues as having been incorporated into the plurality of growing strands;

(c) removing the label and removing by chemical means the chemical moiety of said incorporated nucleotide analogues capping the 3′

-OH group; and

(d) repeating the cycle of steps (a) through (c).

50. The method of claim 49, wherein the DNA polymerase is selected from a group consisting of Taq FS DNA polymerase, T7 DNA polymerase, and Vent (exo-) DNA polymerase.

51. method of claim 49, wherein the unique label is a fluorescent moiety.

52. The method of claim 49, wherein said plurality of deoxyribonucleic acids are able to self prime initiation of the DNA polymerase reaction of step (a).

53. The method of claim 49, wherein the plurality of different deoxyribonucleic acids is covalently immobilized in a plurality of spots on a solid surface, wherein each spot comprises a plurality of the same deoxyribonucleic acid;

and

wherein the unique labels are dyes having a unique fluorescence emission, and the unique fluorescence emission from a specific dye on the dye-labeled nucleotide analogues on each spot of the solid surface will reveal the identity of the incorporated nucleotides.

54. The method of claim 49, further comprising a step of amplifying the different deoxyribonucleic acids before the deoxyribonucleic acids are covalently immobilized.

55. The method of claim 49, wherein at least one of said nucleotide analogues is a 7-deaza adenine or 7-deaza guanine.

56. The method of claim 49, wherein the chemical moiety capping the 3′

-OH group has a steric bulk smaller than a 2-nitrobenzyl moiety.

57. A plurality of different deoxyribonucleic acids covalently immobilized on a solid support, said plurality of different deoxyribonucleic acids comprising incorporated nucleotide analogues, wherein each type of nucleotide analogue is labeled with a unique label attached through a cleavable linker to the base and contains a chemical moiety capping its 3′

-OH group, said chemical moiety being removable by chemical means, and wherein deoxyribonucleic acids having the same sequence are immobilized at a spot and greater than 10,000 spots are present on the solid support.

58. The plurality of different deoxyribonucleic acids of claim 57, wherein each nucleotide analogue comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, or an analogue thereof.

59. The plurality of different deoxyribonucleic acids of claim 57, wherein said deoxyribonucleic acids are able to self prime initiation of a DNA polymerase reaction.

60. The plurality of different deoxyribonucleic acids of claim 57, wherein at least one of said incorporated nucleotide analogues is a 7-deaza adenine or 7-deaza guanine.

61. The plurality of different deoxyribonucleic acids of claim 57, wherein said nucleotide analogues are selected from the group consisting of cytosine, thymine, uracil, deaza-adenine and deaza-guanine and said unique label is attached through a cleavable linker to a 5-position of cytosine, (New) thymine or uracil or to a 7-position of deaza-adenine or deaza-guanine.

62. The plurality of different deoxyribonucleic acids of claim 57, wherein said immobilized deoxyribonucleic acids are spotted at a density of greater than 10,000 spots on a 4 cm×

1 cm chip.

63. The plurality of different deoxyribonucleic acids of claim 57, wherein the chemical moiety capping the 3′

-OH group has a steric bulk smaller than a 2-nitrobenzyl moiety.

64. A method for sequencing a plurality of different deoxyribonucleic acids by synthesis involving the detection of the identity of nucleotide analogues incorporated into a plurality of different growing strands of DNA hybridized to deoxyribonucleic acids, said method comprising:

(a) incorporating one nucleotide analogue into each growing strand of the plurality of different growing strands of DNA using a DNA polymerase reaction, wherein each nucleotide analogue is a terminator in the DNA polymerase reaction and wherein each nucleotide analogue comprises a unique label attached through a cleavable linker to the base and a chemical moiety capping the 3′

-OH group that is removable by chemical means; and

(b) detecting said label of each said incorporated nucleotide analogue, so as to thereby identify 10,000 or more of the incorporated nucleotide analogues, wherein said method is applied simultaneously to different deoxyribonucleic acids covalently immobilized on a solid surface.

65. The method of claim 64, wherein said different deoxyribonucleic acids are able to self prime initiation of said DNA polymerase reaction.

66. method of claim 64, wherein at least one of said nucleotide analogues is a 7-deaza adenine or 7-deaza guanine.

67. The method of claim 64, wherein said nucleotide analogues are selected from the group consisting of cytosine, thymine, uracil, deaza-adenine and deaza-guanine and said unique label is attached through a cleavable linker to a 5-position of cytosine, thymine or uracil or to a 7-position of deaza-adenine or deaza-guanine.

68. The method of claim 64, further comprising prior to step (a), hybridizing a primer to each of the deoxyribonucleic acids.

69. The method of claim 64, wherein each of the deoxyribonucleic acids prior to step (a) contains an azido group.

70. The method of claim 64, wherein a plurality of deoxyribonucleic acids having the same sequence are immobilized at a spot, wherein the spot is one of greater than 10,000 spots on a chip, and wherein the density of spots on the chip is greater than 10,000 spots on a 4 cm×

1 cm chip.

71. The method of claim 64, further comprising a step of amplifying the different deoxyribonucleic acids before the deoxyribonucleic acids are covalently immobilized.

72. The method of claim 64, wherein the chemical moiety capping the 3′

-OH group has a steric bulk smaller than a 2-nitrobenzyl moiety.