Sequencing by incorporation
First Claim
1. A method of sequencing a nucleic acid, the method comprising:
- (i) providing a nucleic acid template and a primer;
(ii) incubating the nucleic acid template and primer with a polymerase and one or more fluorescently labeled nucleotides, thereby adding at least one of the one or more fluorescently labeled nucleotides to the primer, resulting in an extended primer;
(iii) detecting the at least one fluorescently labeled nucleotide added to the primer;
(iv) photobleaching the at least one or the one or more fluoroescently labeled nucleotides on the extended primer by applying a light pulse, wherein photobleaching destroys or reduces the fluorescence of the at least one of the one or more fluorescently labeled nucleotides to an acceptable level without removing the labeled nucleotide(s) from the extended primer.
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Accused Products
Abstract
Nucleotides and nucleotide analogs are used in various sequencing by incorporation/sequencing by synthesis methods. Nucleotide analogs comprising 3′-blocking groups are used to provide reversible chain-termination for sequencing by synthesis. Typical blocking groups include phosphate groups and carbamate groups. Fluorescent nucleotides are used to perform sequencing by synthesis with detection by incorporation of the fluorescently labeled nucleotide, optionally followed by photobleaching and intercalating dyes are used to detect addition of a non-labeled nucleotide in sequencing by synthesis with detection by intercalation. Microfluidic devices, including particle arrays, are used in the sequencing methods.
418 Citations
26 Claims
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1. A method of sequencing a nucleic acid, the method comprising:
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(i) providing a nucleic acid template and a primer;
(ii) incubating the nucleic acid template and primer with a polymerase and one or more fluorescently labeled nucleotides, thereby adding at least one of the one or more fluorescently labeled nucleotides to the primer, resulting in an extended primer;
(iii) detecting the at least one fluorescently labeled nucleotide added to the primer;
(iv) photobleaching the at least one or the one or more fluoroescently labeled nucleotides on the extended primer by applying a light pulse, wherein photobleaching destroys or reduces the fluorescence of the at least one of the one or more fluorescently labeled nucleotides to an acceptable level without removing the labeled nucleotide(s) from the extended 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, 26)
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Specification