Method for the complete chemical synthesis and assembly of genes and genomes

• US 6,521,427 B1
• Filed: 05/12/2000
• Issued: 02/18/2003
• Est. Priority Date: 09/16/1997
• Status: Expired due to Term

• Current Assignee: Centocor Incorporated (Johnson & Johnson)
• Original Assignee: EGEA Biosciences, Inc. (Johnson & Johnson)
• Inventors: Evans, Glen A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Chakrabarti, Arun K.
• Application Number:
  US09/554,929
• Time in Patent Office: 1,012 Days
• Field of Search: 435/6, 435/66, 435/91.1, 435/91.4, 435/194, 536/23.1, 536/24.2, 536/24.3, 536/24.31, 514/675, 514/690, 424/93.2, 800/25, 800/205
• US Class Current: 435/91.1
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00585 Parallel 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 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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 generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms. Thus, the present invention outlines a novel approach to utilizing the results of genomic sequence information by computer directed gene synthesis based on computing on the human genome database. Specifically, the present invention contemplates and describes the chemical synthesis and resynthesis of genes defined by the genome sequence in a host vector and transfer and expression of these sequences into suitable hosts.

Claims

1. A method of synthesizing a double-stranded polynucleotide, comprising:

(a) annealing a 5′

terminal oligonucleotide with a 3′

terminal oligonucleotide of a double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal oligonucleotide of said double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, said next most terminal oligonucleotide having a length of at least about 25 bases, and

(b) repeating step (a) one or more times to sequentially produce a double-stranded polynucleotide.

2. The method of claim 1, further comprising the step of treating said annealed oligonucleotides with a ligating enzyme.

3. The method of claim 1, further comprising the step of amplifying said double-stranded polynucleotide.

4. The method of claim 1, wherein said double-stranded polynucleotide comprises a length selected from the group consisting of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10×

10³, 20×

10³, 30×

10³, 40×

10³, 50×

10³, 60×

10³, 70×

10³, 80×

10³, 90×

10³, 1×

10⁴, 1×

10⁵, 1×

10⁶, 1×

10⁷, 1×

10⁸, 1×

10⁹ and 1×

10¹⁰ base pairs.

5. The method of claim 1, wherein said double-stranded polynucleotide comprises a coding region and a corresponding regulatory element directing the expression of said coding region.

6. The method of claim 5, wherein said regulatory element further comprises a promoter.

7. The method of claim 1, wherein said double-stranded polynucleotide further comprises a plurality of coding regions and a plurality of regulatory elements.

8. The method of claim 7, wherein said coding regions encode products that comprise a biochemical pathway.

9. The method of claim 8, wherein said biochemical pathway is glycolysis.

10. The method of claim 9, wherein said coding regions encode enzymes selected from the group consisting of hexokinase, phosphohexose isomerase, phosphofructokinase-1, aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase.

11. The method of claim 8, wherein said biochemical pathway is lipid synthesis.

12. The method claim 7, wherein said biochemical pathway is cofactor synthesis.

13. The method of claim 7, wherein said pathway involves lipoic acid.

14. The method of claim 7, wherein said biochemical pathway is riboflavin synthesis.

15. The method of claim 7, wherein said biochemical pathway is nucleotide synthesis.

16. The method of claim 15, wherein said nucleotide is a purine.

17. The method of claim 15, wherein said nucleotide is a pyrimidine.

18. The method of claim 7, wherein said coding regions encode enzymes involved in a cellular process selected from the group consisting of cell division, chaperone, detoxification, peptide secretion, energy metabolism, regulatory function, DNA replication, transcription, RNA processing and tRNA modification.

19. The method of claim 18, wherein said energy metabolism is oxidative phosphorylation.

20. The method of claim 1, wherein said double-stranded polynucleotide is a DNA.

21. The method of claim 1, wherein said double-stranded polynucleotide is an RNA.

22. The method of claim 1, wherein said double-stranded polynucleotide is an expression construct.

23. The method of claim 22, wherein said expression construct is a bacterial expression construct.

24. The method of claim 22, wherein said expression construct is a mammalian expression construct.

25. The method of claim 17, wherein said expression construct is a viral expression construct.

26. The method of claim 1, wherein said double-stranded polynucleotide comprises a genome selected from the group consisting of bacterial genome, yeast genome, viral genome, mammalian genome, amphibian genome and avian genome.

27. The method of claim 1, wherein each said overlap is between about 5 base pairs and about 75 base pairs.

28. The method of claim 1, wherein said overlap is selected form the group consisting of about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 30 base pairs, about 35 base pairs, about base pairs, about 45 base pairs, about 50 base pairs, about 55 base pairs, about 60 base pairs, about 65 base pairs, and about 70 base pairs.

29. The method of claim 5, wherein said promoter is selected from the group consisting of CMV IE, SV40 IE, RSV, β

-actin, tetracycline regulatable and ecdysone regulatable.

30. The method of claim 26, wherein said genome is a viral genome.

31. The method of claim 30, wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.

32. The method of claim 1, wherein said double-stranded polynucleotide is a chromosome.

35. The method of claim 1, wherein said double-stranded polynucleotide comprises an artificial chromosome, wherein said chromosome comprises all coding regions and regulatory elements found in a corresponding natural chromosome.

36. The method of claim 35, wherein said corresponding natural chromosome is a human mitochondrial genome.

37. The method of claim 35, wherein said corresponding natural chromosome is a chloroplast genome.

38. The method of claim 1, wherein said double-stranded polynucleotide comprises an artificial genetic system, wherein said system comprises all coding regions and regulatory elements found in a corresponding natural biochemical pathway.

39. The method of claim 38, wherein said biochemical pathway comprises the activities required for glycolysis.

40. The method of claim 38, wherein said biochemical pathway comprises the enzymes required for electron transport.

41. The method of claim 38, wherein said biochemical pathway comprises the enzyme activities required for photosynthesis.

42. The method of claim 1, wherein said next most terminal oligonucleotide of said double-stranded polynucleotide has an overlap of about 50 percent with said first annealed product.

33. A method of producing a viral particle comprising the steps of:

(a) providing a host cell;

(b) transforming said host cell with an artificial viral genome prepared by;

(i) generating a first set of oligonucleotides comprising one strand of said viral genome;

(ii) generating a second set of oligonucleotides comprising the second complementary strand of said viral genome; and

(iii) annealing said first and said second set of oligonucleotides;

wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′

or 3′

end of said viral genome will hybridize with only one complementary oligonucleotide; and

(c) culturing said transformed host cell under conditions such that said viral particle is expressed.

34. The method of claim 33, wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.

43. A method of synthesizing a replication-competent double-stranded polynucleotide, comprising:

(a) annealing a 5′

terminal oligonucleotide with a 3′

terminal oliogonucleotide of a replication-competent double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal oligonucleotide of said replication-competent double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, and

(b) repeating step (a) one or more times to sequentially produce a replication-competent double-stranded polynucleotide.

44. A method of synthesizing a double-stranded polynucleotide, comprising:

(a) annealing a 5′

terminal oligonucleotide with a 3′

terminal oliogonucleotide of a double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, said next most terminal double-stranded polynucleotide comprising at least one oligonucleotide of at least about 25 bases, and

(b) repeating step (a) one or more times to sequentially produce a double-stranded polynucleotide.

45. A method of synthesizing a replication-competent double-stranded polynucleotide, comprising:

(a) annealing a 5′

terminal oligonucleotide with a 3′

terminal oliogonucleotide of a replication-competent double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, and

(b) repeating step (a) one or more times to sequentially produce a replication-competent double-stranded polynucleotide.