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Method of nucleic acid cassette assembly

a nucleic acid cassette and assembly technology, applied in the field of molecular biology, can solve the problems that conventional genetic engineering techniques are limited to allowing manipulation of existing sequences, and achieve the effect of complementing or facilitating the ability of the genom

Inactive Publication Date: 2018-11-29
SYNTHETIC GENOMICS INC
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a synthetic genome that can direct the replication of a cell in which it is located, based on its environmental conditions. The genome can be supplemented with various small molecules and complex components to enhance its ability to replicate. The sequences in the genome provide all the necessary machinery and components to produce a cell and allow it to replicate under specific energy or nutritional conditions. The technical effect of this patent is the creation of a tool that can be used to control the replication of a cell and study its behavior under different conditions.

Problems solved by technology

Conventional genetic engineering techniques are limited to allowing manipulation of existing sequences.

Method used

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  • Method of nucleic acid cassette assembly
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Examples

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example 1

Mycoplasma Comparative Genomics to Identify Genes in a Minimal Gene Set

[0051]Mycoplasma Comparative Genomics. The 13 complete and 2 partial genome sequences currently in the inventors' dataset comprise an in silico laboratory.

[0052]Comparisons of pairs of mycoplasma genomes using the whole genome alignment tool MUMMER shows between species that are closely related such as M. capricolum, M. mycoides SC, and Mesoplasma florum, genome rearrangements are symmetrical about an axis passing through the origins of replication and points that bisect the genome equally. The direction of transcription of rearranged genes almost always stays the same relative to the origin of replication. This phenomenon has been observed for other species bacteria but perhaps never so strikingly as with M. capricolum and M. mycoides SC (see FIG. 2). These reciprocal crossovers suggest that any major removal of DNA from one side of the genome might need to be matched with a similar deletion from the other side ...

example ii

Synthesis of a Mouse Mitochondrial Genome

[0056]The mouse mitochondrial genome is a 16,299 bp circular DNA and its sequence has been critically checked. We designed 682 48-mers to assemble it in three overlapping segments; Si (6,528 bp), S2 (5,328 bp), and S3 (4,508 bp) as diagrammed in FIG. 3A.

[0057]We assembled each of these three pieces by the method described in Smith et al. (2003), supra, modified to reduce the heating damage to the synthetic product. The gel shown in FIG. 3B illustrates products from one such modified procedure that dramatically reduces the time spent at high temperature.

[0058]The synthesis of three overlapping segments comprising the entire mouse mitochondrial genome illustrates that combinations of PCA and PCR can routinely assemble 5-6 Kris Prather segments of DNA and validates our plan to build 5 Kris Prather cassettes for the assembly of a cellular genome.

example iii

Synthesis of 5 kb Cassettes of an Essential Region of the M. Genitaliumgenome

[0059]5 kb cassettes are constructed to generate a synthetic copy of an essential region of the M. genitalium genome—the ribosomal protein genes MG149.1 through MG181. This 18.5 kb region is flanked by genes that tolerate transposon insertions (MG149 and MG182). Sets of 386 top strand and 386 bottom strand oligonucleotides, of 48 nt, were synthesized to cover this region. These nucleic acids are illustrated in FIG. 4.

[0060]The assembly of four overlapping segments (cassettes) comprising these oligonucleotides is performed.

[0061]Using these techniques, cassettes of, for example, 4-6 kb can be constructed that include gene sets of interest (e.g., a minimal genome from a unicellular microorganism), and can be “mixed and matched” with, or altered by substitutions from, e.g., other species, to obtain a custom made genome, which can be introduced into a vesicle or ghost cell for testing, as described above. Synt...

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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. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. patent application Ser. No. 11 / 635,355 filed on Dec. 6, 2006 entitled “Method of Nucleic Acid Cassette Assembly, now issued as U.S. Pat. No. 10,041,060; which claims the benefit and priority from U.S. Provisional Patent Application Ser. No. 60 / 742,542 filed on Dec. 6, 2005, entitled “Synthetic Genomes.” U.S. patent application Ser. No. 11 / 635,355 is related to U.S. Provisional Patent Application Ser. No. 60 / 752,965 filed on Dec. 23, 2005, entitled “Introduction of Genomes into Microorganisms;” U.S. Provisional Patent Application Ser. No. 60 / 741,469 filed on Dec. 2, 2005, entitled “Error Correction Method;” and U.S. Non-Provisional patent application Ser. No. 11 / 502,746 filed on Aug. 11, 2006, entitled “In Vitro Recombination Method,” all of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with gover...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/10C12N15/66
CPCC12N15/66C12N15/10C12N15/1093
Inventor VENTER, J. CRAIGSMITH, HAMILTON O.HUTCHISON, III, CLYDE A.GIBSON, DANIEL G.
Owner SYNTHETIC GENOMICS INC
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