Sequencing by incorporation
First Claim
1. A method of sequencing a nucleic acid, the method comprising:
- (i) hybridizing a nucleic acid template and a primer, producing a hybridized nucleic acid comprising a double-stranded region;
(ii) incubating the hybridized nucleic acid with a polymerase and a series of nucleotides, thereby adding at least one nucleotide to the primer, resulting in an extended double-stranded region, wherein the incubating is performed in the presence of an intercalating dye, which intercalating dye intercalates into the extended double stranded region;
(iii) detecting the intercalating dye, thereby detecting the addition of the at least one nucleotide to the primer; and
,(iv) repeating steps (ii) through (iv) thereby sequencing the nucleic acid.
1 Assignment
0 Petitions
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.
64 Citations
17 Claims
-
1. A method of sequencing a nucleic acid, the method comprising:
-
(i) hybridizing a nucleic acid template and a primer, producing a hybridized nucleic acid comprising a double-stranded region; (ii) incubating the hybridized nucleic acid with a polymerase and a series of nucleotides, thereby adding at least one nucleotide to the primer, resulting in an extended double-stranded region, wherein the incubating is performed in the presence of an intercalating dye, which intercalating dye intercalates into the extended double stranded region; (iii) detecting the intercalating dye, thereby detecting the addition of the at least one nucleotide to the primer; and
,(iv) repeating steps (ii) through (iv) thereby sequencing the nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
-
Specification