Method for Sequencing a Polynucelotide Template
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Abstract
The invention provides methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template.
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Citations
35 Claims
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1-19. -19. (canceled)
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20. A method for sequencing, the method comprising:
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(a) amplifying a template to generate a single-stranded polynucleotide, wherein the single-stranded polynucleotide comprises; a first region of known sequence; a first template region; a second region of known sequence, wherein the first template region is between the first region of known sequence and the second region of known sequence; a second template region; and a third region of known sequence, wherein the second template region is between the second region of known sequence and the third region of known sequence; (b) disposing the single-stranded polynucleotide on a planar support to generate a single-stranded polynucleotide immobilized on the planar support, wherein the single-stranded polynucleotide forms part of a colony comprising identical copies of the single-stranded polynucleotide, the solid support comprising an array of colonies located at discrete sites on the solid support; (c) hybridizing a first primer to the first region of known sequence in the single-stranded polynucleotide immobilized on the planar support; (d) extending the first primer to form a first extended primer hybridized to the single-stranded polynucleotide, wherein the first extended primer comprises a fluorescent label; (e) detecting fluorescence emitted from the fluorescent label in the first extended primer while the first extended primer is hybridized to the single-stranded polynucleotide immobilized on the planar support; (f) denaturing the first extended primer from the single-stranded polynucleotide; (g) hybridizing a second primer to the second region of known sequence in the single-stranded polynucleotide immobilized on the planar support, wherein the second primer is hybridized to the single-stranded polynucleotide after denaturing the first extended primer from the single-stranded polynucleotide; (h) extending the second primer to form a second extended primer hybridized to the single-stranded polynucleotide immobilized on the planar support, wherein the second extended primer comprises a fluorescent label; and (i) detecting fluorescence emitted from the fluorescent label in the second extended primer while the second extended primer is hybridized to the single-stranded polynucleotide. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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Specification