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

• Current Assignee: Blue Heron Biotech, LLC (Eurofins Scientific SE)
• Original Assignee: Blue Heron Biotechnology Incorporated
• Inventors: Parker, Hsing-Yeh, Mulligan, John T.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US10/965,018
• Publication Number:
  US 20050106606A1
• Time in Patent Office: 1,567 Days
• Field of Search: 435/6, 435/91.2, 435/91.15, 435/91.1, 536/25.3, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00698 Measurement and control of process parameters
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Polynucleotides having in excess of 1,000 nucleotides can be prepared using a solid phase synthesis technique. A feature of the technique is the use of a reusable solid support that contains covalently bound oligonucleotide. This covalently bound oligonucleotide is annealed to a bridge oligonucleotide, where the bridge is also annealed to a first oligonucleotide that forms a portion of the target polynucleotide. After the target polynucleotide is synthesized, it can be removed from the solid support under denaturing conditions, and the solid support re-used to prepare additional target polynucleotides. The yield of the target polynucleotide increases when shearing force is applied to the solid support that is linked to the growing oligonucleotide. This shearing force is thought to extend the growing end of the oligonucleotide away from contact with other oligonucleotide bound to the solid support and make that end more accessible to annealing with solution oligonucleotide. The synthesis is conveniently accomplished on a porous frit, where reagents and washing solutions are pumped through the frit.

Claims

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.

2. The method of claim 1 wherein the solution of part (b) is a solution of a partially double-stranded polynucleotide.

3. The method of claim 1 wherein the solution of part (b) passes by the partially double-stranded polynucleotide coupled to the solid support of part (a) at a constant rate of flow.

4. The method of claim 1 wherein the solution of part (b) passes by the partially double-stranded polynucleotide coupled to the solid support of part (a) at a variable rate of flow.

5. The method of claim 1 wherein the solid support is porous.

6. The method of claim 5 wherein the solution of part (b) is required to pass through the pores of the solid support multiple times.