Methods and apparatuses for analyzing polynucleotide sequences
First Claim
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1. A method of analyzing a target polynucleotide comprising:
- (a) providing a primed target polynucleotide attached to a microfabricated synthesis channel;
(b) flowing a first nucleotide through the synthesis channel under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;
(c) determining presence or absence of a signal, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide;
(d) removing or reducing the signal, if present; and
(e) repeating steps (b)-(d) with a further nucleotide, the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer.
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Abstract
Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.
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Citations
54 Claims
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1. A method of analyzing a target polynucleotide comprising:
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(a) providing a primed target polynucleotide attached to a microfabricated synthesis channel;
(b) flowing a first nucleotide through the synthesis channel under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;
(c) determining presence or absence of a signal, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide;
(d) removing or reducing the signal, if present; and
(e) repeating steps (b)-(d) with a further nucleotide, the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of analyzing a target polynucleotide comprising:
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(a) pretreating the surface of a substrate to create surface chemistry that facilitates polynucleotide attachment and sequence analysis;
(b) providing a primed target polynucleotide attached to a surface of a substrate;
(c) providing a labeled first nucleotides to the attached target polynucleotide under conditions whereby the labeled first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;
(d) determining presence or absence of a signal, the presence of a signal indicating that the labeled first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide; and
(e) repeating steps (c)-(d) with a labeled further nucleotide, the same or different from the first labeled nucleotide, whereby the labeled further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33)
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34. A method of analyzing a target polynucleotide comprising:
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(a) providing a primed target polynucleotide;
(b) providing a first nucleotide under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;
wherein a fraction of said first nucleotide is labeled.(c) determining presence or absence of a signal from the primer, the presence of a signal indicating the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide; and
(d) repeating steps (b)-(c) with a further nucleotide, the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer;
wherein a fraction of said further nucleotide is labeled. - View Dependent Claims (35, 36, 37, 38, 39, 40)
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41. An apparatus for analyzing the sequence of a polynucleotide, comprising:
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(a) a flow cell comprising at least one microfabricated synthesis channel; and
(b) an inlet port and an outlet port, said inlet port and outlet port being in fluid communication with said flow cell for flowing fluids into and through said flow cell. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
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Specification