Gene synthesis by convergent assembly of oligonucleotide subsets

• US 9,068,209 B2
• Filed: 05/12/2009
• Issued: 06/30/2015
• Est. Priority Date: 05/14/2008
• Status: Expired due to Fees

• Current Assignee: British Columbia Cancer Agency Branch (Province of British Columbia)
• Original Assignee: British Columbia Cancer Agency Branch (Province of British Columbia)
• Inventors: Coope, Robin, Horspool, Daniel, Holt, Robert A.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US12/991,694
• Publication Number:
  US 20110287490A1
• Time in Patent Office: 2,240 Days
• Field of Search: 435/91.1, 435/91.2
• US Class Current: 1/1
• 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: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase

Abstract

The invention provides a system and method for synthesizing polynucleotides by solid phase assembly oligonucleotide precursors, in accordance with the method, a polynucleotide is partitioned into an ordered set of subunits, wherein each subunit is assembled in a single reaction from a subset of oligonucleotide precursors that uniquely anneal together to produce the subunit. The subunits are then assembled to form the desired polynucleotide. An important feature of the invention is the selection of subunits that are free of undesired sequence elements, such as palindromes, repetitive sequences, and the like, which would result in more than one subunit product alter ligating a pool of oligonucleotide precursors.

Claims

1. A method of synthesizing a polynucleotide on a solid support from a set of oligonucleotide precursors, the method comprising the steps of:

repeatedly partitioning a polynucleotide into candidate subunits and for each candidate subunit generating tilings of oligonucleotide precursors that cover the candidate subunit, the oligonucleotide precursors being selected from a set containing every sequence of a predetermined length;

selecting a partition wherein each candidate subunit has a tiling of oligonucleotide precursors each free of palindromic sequences and comprises a subset of oligonucleotide precursors capable of annealing together to form a unique duplex, thereby producing an ordered set of palindromeless subunits; and

successively ligating the subsets of oligonucleotide precursors of the palindromeless subunits to an initializing duplex with a complementary end in a single reaction to form a unique duplex, the initializing duplex being attached to a solid support, and each subset being ligated in an order corresponding to the ordered set of palindromeless subunits to form the polynucleotide.

2. The method of claim 1 wherein said polynucleotide is releasably attached to said initializing duplex, so that after said polynucleotide is formed, it may be released and separated from said solid support.

3. The method of claim 2 wherein said polynucleotide is released from said solid support by enzymatic cleavage.

4. The method of claim 2 wherein each of said oligonucleotide precursors has a length in the range of from 4 to 14 nucleotides and each of said subsets of oligonucleotide precursors has a size in the range of from 2 to 128 oligonucleotides.

5. The method of claim 1 wherein each palindromeless subunit of said plurality is synthesized on a separate said solid support by ligating said subset of oligonucleotide precursors to said complementary end of said initializing duplex attached thereto.

6. The method of claim 5 wherein said palindromeless subunit is releasably attached to said initializing duplex, so that after said palindromeless subunit is formed such subunit may be released and separated from said solid support.

7. A method of synthesizing a polynucleotide from a set of oligonucleotide precursors, the method comprising the steps of:

repeatedly partitioning a polynucleotide into candidate subunits and for each candidate subunit generating tilings of oligonucleotide precursors that cover the candidate subunit, the oligonucleotide precursors being selected from a set containing every sequence of a predetermined length;

selecting a partition wherein each candidate subunit has a tiling of oligonucleotide precursors each free of palindromic sequences and comprises a subset of oligonucleotide precursors capable of annealing together to form a unique duplex, thereby producing an ordered set of palindromeless subunits, wherein each palindromeless subunit overlaps adjacent palindromeless subunits of the ordered set and the ordered set includes terminal subunits each having a primer binding site releasably attached thereto;

synthesizing each palindromeless subunit of the ordered set on a separate solid support by ligating a subset of oligonucleotide precursors to a complementary end of an initializing duplex attached the solid support in a single reaction to form a unique duplex; and

combining in a polymerase chain reaction the palindromeless subunits from the solid supports and primers specific for the primer binding sites of the terminal subunits so that the palindromeless subunits and primers undergo successive cycles of denaturation and polymerase extension until the polynucleotide is formed.

8. The method of claim 7 wherein each of said palindromeless subunits is releasably attached to said initializing duplex, so that after said palindromeless subunit is formed such subunit may be released and separated from said solid support.

9. The method of claim 7 wherein each of said palindromeless subunit overlaps its adjacent palindromeless subunit in said ordered set so that stable duplexes are capable of forming for said polymerase extension in said polymerase chain reaction.

10. A method of synthesizing a polynucleotide from a set of oligonucleotide precursors, the method comprising the steps of:

(a) repeatedly partitioning a polynucleotide into candidate subunits and for each candidate subunit generating tilings of oligonucleotide precursors that cover the candidate subunit, the oligonucleotide precursors being selected from a set containing every sequence of a predetermined length;

(b) selecting a partition wherein each candidate subunit has a tiling of oligonucleotide precursors each free of palindromic sequences and comprises a subset of oligonucleotide precursors capable of annealing together to form a unique duplex, thereby producing an ordered set of palindromeless subunits;

(c) ligating the oligonucleotide precursors of each subset to an initializing duplex with a complementary end in a single reaction to form a unique duplex comprising a palindromeless subunit that is releasably attached to a solid support;

(d) repeating step (c) until the ordered set of palindromeless subunits is synthesized; and

(e) ligating the ordered set of palindromeless subunits together to form the polynucleotide.

11. The method of claim 10 further including the step of releasing said palindromeless subunit from said solid support after said step of ligating;

and wherein said step (c) of repeating, further includes a step of releasing said palindromeless subunit until said ordered set of palindromeless subunits is synthesized.

12. The method of claim 10 wherein said step of ligating said ordered set of palindromeless subunits includes ligating pairwise said palindromeless subunits to form intermediate ligation products and successively ligating pairwise intermediate ligation products and successive ligation products thereof until said polynucleotide is synthesized.

13. The method of claim 12 wherein said palindromeless subunits are synthesized on separate said solid supports and wherein said intermediate ligation products are formed by ligation of said palindromeless subunits or by ligation of previously synthesized intermediate ligation products.

14. The method of claim 13 wherein said palindromeless subunits are released from said solid support by enzymatic digestion of said initializing duplex.

15. A system for synthesizing a polynucleotide from a set of oligonucleotide precursors, the system comprising:

a source of oligonucleotide precursors, the source capable of providing a set of oligonucleotide precursors of every sequence of a predetermined length;

an ordered set of palindromeless subunits with respect to the set of oligonucleotide precursors, the ordered set of palindromeless subunits covering the polynucleotide, each palindromeless subunit overlapping adjacent palindromeless subunits of the ordered set, the ordered set including terminal subunits each having a primer binding site releasably attached thereto, and each palindromeless subunit comprising a subset of oligonucleotide precursors capable of annealing together to form a unique duplex;

wherein the ordered set of palindromeless subunits is generated by repeatedly partitioning the polynucleotide into candidate subunits and for each candidate subunit generating tilings of oligonucleotide precursors which cover the candidate subunit until a tiling of each candidate subunit is obtained, that comprises a subset of oligonucleotide precursors without palindromic sequences;

a plurality of reaction vessels for synthesizing each palindromeless subunit of the ordered set on a separate solid support by ligating a subset of oligonucleotide precursors to a complementary end of an initializing duplex attached the solid support in a single reaction; and

a second reaction vessel for combining in a polymerase chain reaction the palindromeless subunits from the solid supports and primers specific for the primer binding sites of the terminal subunits so that the palindromeless subunits and primers undergo successive cycles of denaturation and polymerase extension until the polynucleotide is formed.

16. The system of claim 15 wherein said source of oligonucleotide precursors is a bank of reservoirs in fluid communication with said plurality of reaction vessels, each reservoir in the bank containing a different oligonucleotide precursor.

17. The system of claim 15 wherein said source of oligonucleotide precursors is a synthesis station under control of a control system operationally associated with said plurality of reaction vessels, such that the control system directs the parallel synthesis in the synthesis station of each of said oligonucleotide precursors of each of said subsets for each of said palindromeless subunits and successively delivers such oligonucleotide precursors to each of said reaction vessels of said plurality.

18. The system of claim 17 wherein said control system, after assembly of said palindromeless subunits in said plurality of reaction vessels, directs the delivery of said palindromeless subunits to said second reaction vessel and conducts said polymerase chain reaction.