MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA
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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.
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Citations
72 Claims
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1-48. -48. (canceled)
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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:
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(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). - View Dependent Claims (50, 51, 52, 53, 54, 55, 56)
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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.
- View Dependent Claims (58, 59, 60, 61, 62, 63)
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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:
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(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. - View Dependent Claims (65, 66, 67, 68, 69, 70, 71, 72)
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Specification