Methods and compositions for economically synthesizing and assembling long DNA sequences

• US 20030186226A1
• Filed: 03/07/2000
• Published: 10/02/2003
• Est. Priority Date: 03/08/1999
• Status: Abandoned Application

• Current Assignee: Metrigenix Incorporated
• Original Assignee: Metrigenix Incorporated
• Inventors: Brennan, Thomas M., Heyneker, Herbert L.
• Application Number:
  US09/519,927
• Publication Number:
  US 20030186226A1
• 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 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The present invention relates to a cost-effective method of assembling long DNA sequences from short synthetic oligonucleotides. More specifically, short oligonucleotides are synthesized in situ on a solid support and subsequently cleaved from the solid support prior to or during the assembly into the full-length DNA sequences.

Claims

1. A method for producing a DNA sequence of greater than 200 bases long comprising the steps of:

(a) synthesizing on a solid support an array of oligonucleotides from about 10 to 200 bases encoding for either the sense or antisense strand of said DNA sequence wherein said oligonucleotides are covalently attached to the solid support using a cleavable moiety;

(b) cleaving said oligonucleotides from the solid support; and

(c) assembling the oligonucleotides into said DNA sequence.

2. The method according to claim 1 wherein said oligonucleotides are from about 20 to 100 bases long.

3. The method according to claim 1 wherein the length of said DNA sequence ranges from about 200 to 10,000 bases.

4. The method according to claim 1 wherein the length of said DNA sequence ranges from about 400 to 5,000 bases.

5. The method according to claim 1 wherein said cleavable moiety is a succinate like moiety.

6. The method according to claim 1 wherein the number of oligonucleotides synthesized on the solid support is from about 10 to 2000.

7. The method according to claim 1 wherein the number of oligonucleotides synthesized on the solid support is from about 10 to 500.

8. The method according to claim 1 wherein step (c) further comprising enzymatic ligation.

9. The method according to claim 1 wherein step (c) further comprising the use of a DNA polymerase.

10. The method according to claim 1 wherein step (c) further comprising the use of a restriction enzyme.

11. The method according to claim 1 wherein said DNA sequence encodes a gene.

12. The method according to claim 1 wherein said DNA sequence is a plasmid.

13. The method according to claim 1 wherein said DNA sequence is a virus.

14. The method according to claim 1 wherein said DNA sequence is the genome of an organism.

15. A DNA of greater than 200 bases long recovered according to the method of claim 1.

16. A solid support containing a cleavable moiety for oligonucleotide synthesis according to the method of claim 1.

18. The DNA sequence produced according to claim 1.

19. The solid support prepared according to claim 1(a).

17. A method for optimizing the function of a DNA sequence comprising the steps of:

a) synthesizing on a solid support an array of oligonucleotides from 10 to 200 bases encoding for either the sense or antisense strand of said DNA sequence wherein said oligonucleotides are covalently attached to the solid support using a cleavable moiety;

(b) cleaving said oligonucleotides from the solid support;

(c) assembling the oligonucleotides into said DNA sequence.

(d) testing the function of said DNA sequence; and

(e) repeating the steps of (a)-(d) by varying said DNA sequence to optimize the function.