Method for sequencing a polynucleotide template

摘要

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.

主权项

1. A method for sequencing, the method comprising:
(a) providing a single-stranded polynucleotide immobilized on a planar support, 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; anda 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) hybridizing a first primer to the first region of known sequence in the single-stranded polynucleotide immobilized on the planar support; (c) 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; (d) 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; (e) denaturing the first extended primer from the single-stranded polynucleotide; (f) 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; (g) 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 (h) detecting fluorescence emitted from the fluorescent label in the second extended primer while the second extended primer is hybridized to the single-stranded polynucleotide.