Method of nucleic acid cassette assembly

• US 10,041,060 B2
• Filed: 12/06/2006
• Issued: 08/07/2018
• Est. Priority Date: 12/06/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Venter, J. Craig, Smith, Hamilton O., Hutchison, III, Clyde A., Gibson, Daniel G.
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US11/635,355
• Publication Number:
  US 20070264688A1
• Time in Patent Office: 4,262 Days
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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

Abstract

Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

Claims

1. A method for assembling a nucleic acid construct comprising:

(a) generating a plurality of double-stranded nucleic acid cassettes from chemically synthesized oligonucleotides or copies thereof,

wherein the cassettes comprise adjacent regions of a nucleic acid construct to be assembled and wherein each of the cassettes overlap with one or more other cassette(s) by at least 50 nucleotides and is between about 4 kb and 7 kb in length, not including the overlaps; and

(b) simultaneously assembling the plurality of nucleic acid cassettes in vitro in a chew back and repair step utilizing;

an enzyme having 3′

or 5′

exonuclease activity,

a DNA polymerase,

polyethylene glycol (PEG) or a single-stranded binding protein, and

a ligase

thereby assembling the nucleic acid construct.

2. The method of claim 1 wherein the enzyme having exonuclease activity is a 5′

exonuclease and the step (b) utilizes PEG.

3. The method of claim 2 wherein the assembly is performed in a single step.

4. The method of claim 2 wherein the plurality of nucleic acid cassettes comprises more than 4 nucleic acid cassettes.

5. The method of claim 4 wherein the plurality of nucleic acid cassettes comprises more than 6 nucleic acid cassettes.

6. The method of claim 2, wherein the cassettes are from about 4.5 kilobases to about 6.5 kilobases in length, not including overlaps.

7. The method of claim 2, wherein the cassettes are about 5 kilo bases in length, not including overlaps.

8. The method of claim 2, wherein each of the cassettes overlaps an adjacent cassette by at least 200 nucleotides.

9. The method of claim 1, wherein the assembled nucleic acid construct is a non-naturally occurring genome nucleic acid construct.

10. The method of claim 1, wherein one or more of the cassettes includes restriction enzyme sites or adaptor sites.

11. The method of claim 1, wherein each cassette is generated entirely from chemically synthesized oligonucleotides or copies thereof.

12. The method of claim 2, wherein the method is automated.

13. The method of claim 1, wherein generating the cassettes comprises ligating the chemically synthesized oligonucleotides to form ligation products and performing cycles of polymerase cycle assembly (PCA) on the ligation products, thereby forming PCA products.

14. The method of claim 13, wherein the PCA consists of 5 cycles of PCA and the ligation is performed at 50°

C.

15. The method of claim 1, wherein step (a) further comprises cloning or amplifying the cassettes.

16. The method of claim 1, wherein the chemically synthesized oligonucleotides or copies thereof are between about 30 nucleotides and 1 kilo base in length.

17. The method of claim 1, wherein each of the cassettes overlaps an adjacent cassette by about 50-1300 nucleotides.

18. The method of claim 1 wherein the plurality of nucleic acid cassettes comprises at least 4 nucleic acid cassettes.

19. The method of claim 18 wherein the plurality of nucleic acid cassettes comprises more than 6 nucleic acid cassettes.

20. The method of claim 1, wherein the cassettes are from about 4.5 kilobases to about 6.5 kilobases in length, not including overlaps.

21. The method of claim 1, wherein each of the cassettes overlaps an adjacent cassette by at least 200 nucleotides.