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Solid phase methods for polynucleotide production

  • US 7,482,119 B2
  • Filed: 10/13/2004
  • Issued: 01/27/2009
  • Est. Priority Date: 04/01/2002
  • Status: Expired due to Term
First Claim
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1. A method of preparing a double-stranded polynucleotide product in solution, comprising the following steps:

  • (a) providing a partially double-stranded polynucleotide coupled to a solid support, wherein the partially double-stranded polynucleotide coupled to the solid support comprises an universal oligonucleotide and a bridge oligonucleotide, wherein one end of the universal oligonucleotide is coupled to the solid support and the other end of the universal oligonucleotide is non-reactive under ligating conditions and wherein the universal oligonucleotide is annealed to a portion of the bridge oligonucleotide;

    (b) contacting a solution of a first single-stranded polynucleotide or a first partially double-stranded polynucleotide, both of which are at least partially complementary to the portion of the bridge oligonucleotide that is not annealed to the universal oligonucleotide, with the partially double-stranded polynucleotide coupled to the solid support of part (a);

    (c) passing the solution of part (b) in one direction by the partially double-stranded polynucleotide coupled to the solid support of part (a) at a rate of flow to allow for the single-stranded polynucleotide or the partially double-stranded polynucleotide in the solution of part (b) to anneal to the portion of the bridge oligonucleotide that is not annealed to the universal oligonucleotide;

    (d) passing the solution of part (b) in an opposite direction from the previous direction by the partially double-stranded polynucleotide coupled to the solid support of part (a) at a rate of flow in order to allow for the first single-stranded polynucleotide or the first partially double-stranded polynucleotide in the solution of part (b) to anneal to the portion of the bridge oligonucleotide that is not annealed to the universal oligonucleotide; and

    (e) repeating part (d) multiple times as needed to form a first intermediate duplex coupled to the solid support wherein at least 80% of the first single-stranded polynucleotide or the first partially double-stranded polynucleotide in the solution of part (c) anneals to the portion of the bridge oligonucleotide that is not annealed to the universal oligonucleotide and wherein the first intermediate duplex comprises a nucleotide region that is not annealed to the bridge oligonucleotide;

    (f) contacting the first intermediate duplex with a solution of a second single-stranded polynucleotide or a second partially double-stranded polynucleotide, both of which are at least partially complementary to the nucleotide region of the first intermediate duplex that is not annealed to the bridge oligonucleotide;

    (g) passing the solution of part (d) in one direction by the first intermediate duplex at a rate of flow to allow for the second single-stranded polynucleotide or the second partially double-stranded polynucleotide in the solution of part (f) to anneal to the first intermediate duplex;

    (h) passing the solution of part (f) in an opposite direction from the previous direction by the first intermediate duplex at a rate of flow in order to allow for the second single-stranded polynucleotide or the second partially double-stranded polynucleotide in the solution of part (f) to anneal to first intermediate duplex; and

    (i) repeating part (h) multiple times as needed to form a second intermediate duplex wherein at least 80% of the second single-stranded polynucleotide or the second partially double-stranded polynucleotide in the solution of part (f) anneals to the nucleotide region of the first intermediate duplex that is not annealed to the bridge oligonucleotide and wherein the second intermediate duplex comprises a nucleotide region that is not annealed to the first intermediate duplex;

    (j) repeating steps (b) through (e) as needed to form a final annealed polynucleotide product coupled to the solid support;

    (k) exposing the final annealed polynucleotide product to ligating conditions and denaturing conditions to provide the double-stranded polynucleotide product, wherein the universal oligonucleotide does not form part of the double-stranded polynucleotide product.

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