Solid phase methods for polynucleotide production

• US 20050106606A1
• Filed: 10/13/2004
• Published: 05/19/2005
• Est. Priority Date: 04/01/2002
• Status: Active Grant

• Current Assignee: Blue Heron Biotech, LLC (Eurofins Scientific SE)
• Original Assignee: Blue Heron Biotechnology Incorporated
• Inventors: Parker, Hsing-Yeh, Mulligan, John T.
• Granted Patent: US 7,482,119 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 for gene or gene fragment assembly, comprising the following steps:

(a) providing a partially double-stranded polynucleotide coupled to a solid support;

(b) providing a solution of a single-stranded or a partially double-stranded polynucleotide that is at least partially complementary to a single stranded portion of the partially double-stranded polynucleotide of step (a);

(c) contacting the solid support of step (a) with the solution of step (b) under the influence of a force exerted in one direction where at least some of the solution of step (b) passes by the partially double-stranded polynucleotide of step (a); and

(d) reversing the direction of the force exerted in step (c) at least once so that at least some of the solution of step (b) passes by the partially double-stranded polynucleotide of step (a) at least twice;

wherein the single-stranded or partially double-stranded polynucleotide of step (b) anneals to the single-stranded portion of the partially double-stranded polynucleotide of step (a).

2. The method of claim 1 wherein the direction of the force exerted in step (c) is reversed multiple times so that the partially double-stranded polynucleotide of step (a) is repetitively contacted with the solution of step (b).

3. The method of claims 2 or 3 wherein the single-stranded or partially double-stranded polynucleotide of step (b) is partially double-stranded.

6. The method of claim 1 or 4 wherein the force is exerted at a constant level.

7. The method of claim 1 or 4 wherein the force is exerted at a variable level.

8. The method of claims 1 or 4 wherein the solid support is porous.

9. The method of claim 8 wherein the solution of step (b) is required to pass through the pores of the solid support multiple times.

4. A method for gene or gene fragment assembly, comprising the following steps:

(a) providing a partially double-stranded polynucleotide coupled to a solid support;

(b) providing a solution of a single-stranded or a partially double-stranded polynucleotide that is at least partially complementary to a single stranded portion of the partially double-stranded polynucleotide of step (a); and

(c) contacting the solid support of step (a) with the solution of step (b) under the influence of a force exerted in a direction where at least some of the solution of step (b) passes by the partially double-stranded polynucleotide of step (a);

wherein the single-stranded or partially double-stranded polynucleotide of step (b) anneals to the single-stranded portion of the partially double-stranded polynucleotide of step (a).

5. The method of claim 4 wherein the single-stranded or partially double-stranded polynucleotide of step (b) is partially double-stranded.

10. A device for automated gene fragment or gene assembly, comprising:

(a) a reaction block comprising a plurality of cavities adapted to hold a plurality of reaction vessels, wherein each reaction vessel, when present within a cavity, comprises a first orifice, a second orifice, and a solid support positioned within the interior of the reaction vessel between the first and second orifice; and

(b) a reagent delivery and mixing unit in fluid communication with the plurality of reaction vessels when present.

11. The device of claim 10 wherein the reaction block further comprises one or more means to control or monitor the temperature of the reaction block.

12. The device of claim 10 wherein the reaction block further comprises one or more means to monitor the fluid level within a reaction vessel when present.

13. The device of claim 10 further comprising one or more means to immobilize the plurality of reaction vessels within the plurality of the cavities of the reaction block.

14. The device of claim 10 wherein the reagent delivery and mixing unit contains one or more pumps in fluid communication with one or more valves.

15. The device of claim 14 wherein a valve of the reagent delivery and mixing unit is positioned between the first orifice of a reaction vessel when present and one of the pumps of the reagent delivery and mixing unit, where the valve is in fluid communication with the pump, the reaction vessel and one or more liquid storage containers.

16. The device of claim 15 where a liquid in one of the liquid storage containers is transported from the liquid storage container through the valve into the reaction vessel by the action of the pump.

17. The device of claim 10 further comprising a microplate holding apparatus.

18. The device of claim 17 wherein the microplate holding apparatus further comprises a temperature monitoring and control means.

19. The device of claim 17 further comprising a multi-microplate storage system.

20. The device of claim 19 wherein the multi-microplate storage system further comprises a temperature monitoring and control means.

21. The device of claim 20 further comprising a means to transport a microplate from the multi-microplate storage system to the microplate holding apparatus.

22. The device of claim 17 further comprising a microplate-well seal-piercing means.

23. The device of claim 10 further comprising a barcode reader.

24. The device of claim 10 further comprising one or more means to analyze the products of the processes performed by the device.

25. The device of claim 24 where the means is a spectrophotometer.

26. The device of claim 24 where the means is a fluorescence plate reader.

27. The device of claim 10 further comprising a computer control unit, where the computer control unit executes pre-programmed commands to operate the device.

28. The device of claim 27 wherein the computer control unit executes pre-programmed commands to operate the action of reagent delivery and mixing unit.

29. The device of claims 11, 18, and 20 wherein the computer control unit executes pre-programmed commands to operate the temperature monitor and control means.

30. The device of claim 12 wherein the computer control unit executes pre-programmed commands to operate the fluid monitor means of the reaction block.

31. The device of claim 21 wherein the computer control unit executes pre-programmed commands to operate the means to transport a microplate from the multi-microplate storage system to the microplate holding apparatus.

32. The device of claim 22 wherein the computer control unit executes pre-programmed commands to operate the microplate-well seal-piercing means.

33. A device for automated gene fragment or gene assembly, comprising:

(a) a reaction block comprising a plurality of cavities adapted to hold a plurality of reaction vessels, wherein each reaction vessel, when present within a cavity, comprises a first orifice, a second orifice, and a solid support positioned within the interior of the reaction vessel between the first and second orifice;

(b) a reagent delivery and mixing unit in fluid communication with the plurality of reaction. vessels when present;

(c) a microplate holding apparatus;

(d) one or more means to immobilize the plurality of reaction vessels when present within the plurality of cavities of the reaction block;

(e) one or more means to monitor or control the temperature of the reaction block, the multi-microplate storage system or the microplate holding apparatus;

(f) a computer control unit, where the computer control unit executes pre-programmed commands to operate the device;

(g) optionally, a multi-microplate storage system;

(h) optionally, a means to transport a microplate from a multi-microplate storage system to a microplate holding area; and

(i) optionally, a microplate well seal-piercing means.