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DNA sequencing using controlled strand displacement

  • US 10,227,647 B2
  • Filed: 02/10/2016
  • Issued: 03/12/2019
  • Est. Priority Date: 02/17/2015
  • Status: Active Grant
First Claim
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1. A method of producing a DNA strand for sequencing, comprisinga) providing a template DNA polynucleotide strand comprising a first target DNA sequence interposed between a first adaptor 3′

  • to the first target DNA sequence and a second adaptor 5′

    to the first target DNA sequence, wherein the template DNA polynucleotide strand is immobilized on a substrate and is provided as a first strand,b) combining a first primer with the immobilized template DNA polynucleotide strand, and hybridizing the first primer to a first primer binding sequence in the first adaptor, wherein the first primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide strand;

    c) extending the first primer using one or more DNA polymerases to generate a second strand, wherein the second strand comprises a sequence complementary to the first target DNA sequence and a sequence complementary to at least part of the second adaptor;

    d) combining a second primer with the immobilized template DNA polynucleotide strand, hybridizing a second primer to a second primer binding sequence in the first adaptor, wherein the second primer binding sequence is 3′

    to the first primer binding sequence, wherein the second primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide strand; and

    e) extending the second primer using a DNA polymerase having strand-displacement activity to generate a third strand,wherein extending the second primer to generate the third strand is carried out under conditions in which the extension of the second primer partially displaces the second strand, thereby producing a partially hybridized second strand having;

    (i) a hybridized portion that is hybridized to the template DNA polynucleotide, and(ii) an unhybridized portion that contains a sequence that is complementary to the first target DNA sequence and a sequence that is complementary to at least part of the second adaptor, wherein the unhybridized portion is 5′

    in the second strand sequence to the hybridized portion.

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