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Method for sequencing a polynucelotide template

  • US 9,994,896 B2
  • Filed: 12/17/2014
  • Issued: 06/12/2018
  • Est. Priority Date: 02/08/2006
  • Status: Active Grant
First Claim
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1. A method for sequencing, the method comprising:

  • (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.

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