Multiple reactor system and method for polynucleotide synthesis

• US 4,517,338 A
• Filed: 07/13/1984
• Issued: 05/14/1985
• Est. Priority Date: 06/20/1983
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Urdea, Mickey S., Warner, Brian D.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Nutter, Nathan M.
• Application Number:
  US06/630,781
• Time in Patent Office: 305 Days
• Field of Search: 435/87, 435/88, 435/91, 435/172, 435/287, 435/317, 435/820, 525/54.1, 525/54.11, 525/54.23, 935/88, 422/116, 422/131
• US Class Current: 536/25.3
• 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 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00686 Automatic
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y10S 435/82 Subcellular parts of microorganisms

Abstract

A reactor system and method for synthesizing or degrading polynucleotides and other linear polymers includes a tubular reactor connected to a reagent manifold. The polynucleotide is immobilized on a loosely packed solid-phase support material in the tubular reactor, and reagents are sequentially introduced into the tubular reactor. After each reagent is introduced, the tubular reactor is isolated from the reagent manifold and the reagent agitated by alternately pressurizing the opposite ends of the tubular reactor. The method provides rapid and efficient synthesis of polynucleotides. By connecting two or more tubular reactors to the reagent manifold, a plurality of polynucleotides having different sequences may be synthesized simultaneously.

Claims

1. A reactor system for the sequential modification of two more linear polymers attached to dispersed solid-phase support, said reactor system comprising:

a plurality of reactor zones for holding said dispersed solid-phase supports, said reactor zones having at least two access ports defined by porous barriers;

a reagent manifold for selectively delivering particular reagents to each reactor zone one at a time through said porous barriers;

means for selectively blocking the flow of reagents to the reactor zones from the reagent manifold; and

means for selectively inducing a continuously reversing flow of reagent through each reactor zone when the flow of reagents to that reactor is blocked.

2. A reactor system as in claim 1, wherein each reactor zone is defined by an elongated tubular column which is open at both ends, said open ends defining the access ports and having the porous barriers thereacross.

3. A reactor system as in claim 1, further including a surge volume between the means for blocking and the ends of each reactor zone.

4. A reactor system as in claim 1, wherein the means for inducing a continuously reversing flow of reagents includes means for supplying a pressurized inert gas and means for directing said pressurized inert gas alternately to both of said access ports on each reactor zone.

5. A reactor system as in claim 4, wherein the means for directing said pressurized inert gas comprises a three-way valve intermediate the inert gas supply means and each access port of the reactor zone.

6. A reactor system as in claim 1, wherein the means for blocking includes first and second isolation valves capable of selectively directing reagents to each reactor.

7. A reactor system as in claim 5, wherein the means for directing further include surge volume between each three-way valve and the reactor zone, said surge volume being at least equal to the volume of the reactor zone.

8. A reactor system for the sequential modification of two more linear polymers attached to dispersed solid-phase supports, said system comprising:

a plurality of tubular reactors for holding said solid-phase supports, said reactor being enclosed at each end by a porous barrier;

a plurality of reagent reservoirs and wash reservoirs, at least some of said reagent and wash reservoirs being arranged in pairs;

a reagent manifold having a plurality of entry ports for receiving reagents and wash solutions from said reservoirs, and at least one outlet for directing said reagents and wash solutions to the reactors, wherein said pairs of reagents and wash solutions each enter the manifold through common entry ports; and

means for selectively blocking each reactor from the reagent manifold; and

means for alternately pressurizing the two opposite ends of each reactor to induce a reversing flow of reagents or wash solutions therethrough when the reagent manifold is blocked.

9. A reactor system as in claim 8, wherein the pairs of reagents and wash solutions are connected to their respective entry ports through common valves to assure that the reagent is completely washed from the manifold.

10. A reactor system as in claim 9, wherein each reagent and wash solution pair is connected to the manifold through two valves in series.

11. A reactor system as in claim 10, wherein the two valves in series include a two-way common port valve connected to the entry port in the reagent manifold and a three-way valve connected to selectively direct either reagent or wash solution to the two-way valve.

12. A reactor system as in claim 8, wherein the reagent and wash reservoirs are capable of being pressurized to induce a flow of reagent and wash solutions through the manifold to the reactor zone.

13. A reactor system as in claim 8, further comprising a control system for selectively directing reagent and wash solutions to the reactor zones, blocking the reactor zones, and pressurizing the reactor zones to simultaneously induce a reversing flow of reagents or wash solutions therethrough.

14. A reactor system as in claim 12, further including means for selectively pressurizing the reagents and wash reservoirs to deliver reagent and wash solutions to the reactor zone through the reagent manifold.

15. A reactor system as in claim 14, wherein the pressurizing means further includes means for isolating the reservoirs from each other to prevent cross contamination.

16. A method for simultaneously synthesizing two or more polynucleotides by selectively introducing reagents to a plurality of solid-phase substrates, each substrate being contained in a tubular reactor zone, whereby individual nucleotides are sequentially added to a chain of nucleotides attached to each of said substrates, said method characterized by retaining at least some of the reagents within each reactor zone and simultaneously inducing an oscillating flow of reagents by alternately pressurizing both sides of each reactor.

17. A method for synthesizing polynucleotides as in claim 16, wherein the flow of reactants in each reactor zone is reversed at a frequency from 1 to 10 Hz.

18. A method for synthesizing polynucleotides as in claim 16, wherein the oscillating flow of reagents is induced by alternately pressurizing each side of the reactors, with an inert gas.

19. A method as in claim 18, wherein the inert gas is helium.