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Preparations, compositions, and methods for nucleic acid sequencing

  • US 8,795,961 B2
  • Filed: 09/03/2009
  • Issued: 08/05/2014
  • Est. Priority Date: 09/05/2008
  • Status: Active Grant
First Claim
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1. A method of sequencing nucleic acids, comprising;

  • a) providing a sample comprising target nucleic acids;

    b) adding a set of primers to said sample, wherein the set comprises primers having different nucleotide compositions;

    c) contacting the target nucleic acids in the sample with the set of primers under conditions that promote hybridization to generate a set of target-primer complexes;

    d) using a polymerase enzyme, pre-extending primers in the target-primer complexes to generate pre-extended target-primer complexes that further comprise the polymerase enzyme;

    e) confining each of the pre-extended target-primer complexes to a separate reaction site on a substrate comprising a plurality of reaction sites;

    f) initiating template-directed synthesis of nascent nucleic acid strands from each of the pre-extended target-primer complexes, wherein the nascent nucleic acid strands are complementary to the target nucleic acids, and wherein the template-directed synthesis is performed by the polymerase enzyme;

    g) detecting processive incorporation of nucleotides into each of the nascent nucleic acid strands with no intervening buffer exchange during the template-directed synthesis, wherein the detecting further identifies nucleotide sequence reads for the nascent nucleic acid strands; and

    h) based upon the results of step f, determining a nucleotide sequence of the target nucleic acids.

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