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Low sequence bias single-stranded DNA ligation

  • US 9,816,120 B2
  • Filed: 01/09/2014
  • Issued: 11/14/2017
  • Est. Priority Date: 01/09/2013
  • Status: Active Grant
First Claim
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1. A method of producing a ligated single stranded nucleic acid molecule, the method comprising:

  • a) contacting a single-stranded acceptor nucleic acid molecule with a population of donor nucleic acid molecules, wherein each of the donor nucleic acid molecules comprises a stem region, and a single-stranded 3′

    terminal overhang region, wherein the sequence of the donor nucleic acid is designed to contain at least one mismatch internally in the stem region, and wherein at least the four 5′

    terminal nucleotides of the donor molecule form Watson-Crick base pairs as part of the stem region, and wherein the donor molecule comprises at least one randomized templating nucleotide in the 3′

    terminal overhang region at the position nearest the stem loop, thereby allowing unbiased hybridization with a multiplicity of different acceptor molecules;

    b) hybridizing the single stranded 3′

    terminal overhang region of the donor nucleic acid molecule to the acceptor molecule under conditions where the stem structure is formed in the donor molecule, wherein the acceptor molecule may comprise any nucleotide sequence at the 3′

    end, thereby forming an acceptor-donor hybrid molecule comprising a nick or gap between the acceptor nucleic acid and donor nucleic acid molecule; and

    c) ligating the 5′

    end of the donor nucleic acid molecule to the 3′

    end of the acceptor nucleic acid molecule with a ligase that is active under conditions where the stem structure is formed in the donor nucleic acid molecule, thereby generating a ligated product, whereby the yield of ligated product is at least 65%.

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