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Method of preparing libraries of template polynucleotides

  • US 8,563,478 B2
  • Filed: 04/20/2010
  • Issued: 10/22/2013
  • Est. Priority Date: 11/01/2005
  • Status: Active Grant
First Claim
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1. A method of generating a library of different polynucleotide molecules, the method comprising:

  • (a) providing a plurality of different target polynucleotide duplexes;

    (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region;

    (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and(d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs,wherein the extension products comprise nucleic acid sequences that differ from the nucleic acid sequences of either strand of the adapter-target constructs and the extension products collectively provide a library of different template polynucleotide molecules which have common sequences at their 5′

    ends and common sequences at their 3′

    ends;

    wherein the polynucleotide adapters are forked adapters formed by annealing of partially complementary first and second polynucleotide strands; and

    wherein a sequence of 5 or more consecutive nucleotides at 3′

    end of the first strand of the forked adapters is complementary to a sequence of 5 or more consecutive nucleotides the 5′

    end of the second strand of the forked adapters such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands.

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