Method for the enzymatic synthesis of oligonucleotides using thermostable 3'-phosphatase

• US 5,629,177 A
• Filed: 06/13/1994
• Issued: 05/13/1997
• Est. Priority Date: 12/23/1992
• Status: Expired due to Fees

• Current Assignee: Hyman, Edward D.
• Original Assignee: Hyman, Edward D.
• Inventors: Hyman, Edward D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/259,308
• Time in Patent Office: 1,065 Days
• Field of Search: 435/91, 435/6, 536/25.3, 536/25.6, 935/16
• US Class Current: 435/91.2
• 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 2219/00283 Reactor vessels with top opening
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00481 by the use of moving stirrers within the reaction vessels
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• 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
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/101 : Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2535/113 : Cycle sequencing
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/08 : Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials

Abstract

An oligonucleotide is synthesized by adding a 3'"'"'-phosphate blocked nucleotide to a primer, removing the blocking group from the primer-blocked nucleotide product using a thermostable 3'"'"'-phosphatase enzyme, and repeating these steps until the desired nucleotides have been added to the primer. A suitable phosphatase enzyme for use in this method is a thermostable phosphatase derived from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Claims

1. A method for synthesizing a portion of an oligonucleotide of defined sequence, comprising the steps of:

(a) combining an oligonucleotide primer and a blocked nucleotide, or a blocked nucleotide precursor that forms a blocked nucleotide substrate in situ, in a reaction mixture in the presence of RNA Ligase, such that a primer-blocked nucleotide product is formed, wherein the blocked substrate comprises a nucleotide to be added to form part of the defined sequence and a 3'"'"'-phosphate blocking group attached to the nucleotide effective to prevent the addition of more than one blocked substrate to the oligonucleotide primer;

(b) removing the blocking group from the 3'"'"' end of the primer-blocked nucleotide product using a thermostable 3'"'"'-Phosphatase to form a primer-nucleotide product; and

(c) repeating a cycle of steps (a) and (b) using the primer-nucleotide product from step (b) as the oligonucleotide primer of step (a) of the next cycle, without prior purification of the primer-nucleotide product from the reaction mixture.

2. A method according to claim 1, wherein step (c) is performed without replenishment of 3'"'"'-Phosphatase.

3. A method according to claim 2, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

4. A method according to claim 3, wherein the archaebacterium is Pyrococcus furiosus.

5. A method according to claim 2, wherein the blocked substrate is AppNp, where N represents a nucleoside or nucleoside analog, such that RNA Ligase can couple the AppNp to the oligonucleotide primer and such that primer-pN product is able to serve as the primer in the next cycle.

6. A method according to claim 5, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

7. A method according to claim 6, wherein the archaebacterium is Pyrococcus furiosus.

8. A method according to claim 5, wherein step (c) is performed without replenishment of the blocked substrate.

9. A method according to claim 8, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

10. A method according to claim 9, wherein the archaebacterium is Pyrococcus furiosus.

11. A method according to claim 5 further comprising the step of inactivating unreacted blocked nucleotide in the reaction mixture using a Dinucleotide Pyrophosphate Degrading Enzyme prior to step (c) to render it less reactive as a substrate for the chain extending enzyme.

12. A method according to claim 11, wherein the Dinucleotide Pyrophosphate Degrading Enzyme is Nucleotide Pyrophosphatase.

13. A method according to claim 12, wherein the Nucleotide Pyrophosphatase is derived from snake venom.

14. A method according to claim 1, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

15. A method according to claim 14, wherein the archaebacterium is Pyrococcus furiosus.

16. A method according to claim 1, wherein the blocked substrate is AppNp, where N represents a nucleoside or nucleoside analog, such that RNA Ligase can couple the AppNp to the oligonucleotide primer and such that primer-pN product is able to serve as the primer in the next cycle.

17. A method according to claim 16, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

18. A method according to claim 17, wherein the archaebacterium is Pyrococcus furiosus.

19. A method according to claim 16, wherein step (c) is performed without replenishment of the blocked substrate.

20. A method according to claim 19, wherein the 3'"'"'-Phosphatase is derived from a thermophilic archaebacterium.

21. A method according to claim 20, wherein the archaebacterium is Pyrococcus furiosus.

22. A method according to claim 16, further comprising the step of inactivating unreacted blocked nucleotide in the reaction mixture using a Dinucleotide Pyrophosphate Degrading Enzyme prior to step (c) to render it substantially less reactive as a substrate for the chain extending enzyme.

23. A method according to claim 22, wherein the Dinucleotide Pyrophosphate Degrading Enzyme is Nucleotide Pyrophosphatase.

24. A method according to claim 23, wherein the Nucleotide Pyrophosphatase is derived from snake venom.