Methods for in vitro joining and combinatorial assembly of nucleic acid molecules
Inventors
Gibson, Daniel G. • Smith, Hamilton O. • HUTCHISON, Clyde A. • Young, Lei • Venter, J. Craig
Assignees
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Publication Number
US-12371724-B2
Publication Date
2025-07-29
Expiration Date
Abstract
The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.
Core Innovation
The invention provides an in vitro method of joining a set of two or more double-stranded or single-stranded DNA molecules by contacting the molecules in a single vessel in a one-step reaction. Adjacent DNA molecules contain overlapping sequences at their termini, and the joining is carried out without restriction enzymes under conditions effective for joining to form a first assembled dsDNA molecule.
The joining process is implemented with isolated non-thermostable exonuclease and thermostable DNA polymerase components together with a thermostable ligase, plus a crowding agent, a mixture of dNTPs, and a suitable buffer. In one form the method uses a non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity and an isolated thermostable non-strand-displacing DNA polymerase with 3′ exonuclease activity, or a mixture with a second DNA polymerase that lacks 3′ exonuclease activity.
In an alternative form, the invention uses a non-thermostable 3′ to 5′ exonuclease active in the presence of dNTPs, a crowding agent, a heat-activated DNA polymerase, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer to form a first assembled dsDNA molecule in a one-step thermocycled reaction. The invention also provides methods of modifying the properties of a whole nucleic acid molecule by representationally dividing the nucleic acid sequence into portions, identifying sequences of partial nucleic acid molecules, providing a multiplicity of variants for at least a specified number of partial nucleic acid molecules, combinatorially assembling the variants with unvaried partial nucleic acid molecules using overlapping termini, and expressing the variants to determine modified properties.
Claims Coverage
The independent claims cover overlap-based in vitro joining of two or more DNA molecules in a single vessel using specified exonuclease/polymerase/ligase ensembles under isothermal or thermocycled conditions, and representationally dividing and combinatorially assembling whole nucleic acid sequence variants using overlapping termini followed by expression to determine modified properties. The claims identify six inventive features.
Single-vessel one-step overlap-based DNA joining with 5′-to-3′ exonuclease and thermostable polymerase
An in vitro method of joining a set of two or more ds or ss DNA molecules in a single vessel by contacting the DNA molecules with an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity, a crowding agent, an isolated thermostable non-strand-displacing DNA polymerase with 3′ exonuclease activity or a mixture with a second DNA polymerase that lacks 3′ exonuclease activity, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer under conditions effective for joining the DNA molecules to form a first assembled dsDNA molecule in a one-step reaction.
One-step isothermal overlap-based DNA joining kit
A kit for a one-step in vitro reaction to join a set of two or more ds or ss DNA molecules with adjacent molecules containing overlapping sequences at their termini, comprising in a single vessel an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity, a crowding agent, an isolated thermostable non-strand-displacing DNA polymerase with 3′ exonuclease activity or a mixture with a second DNA polymerase that lacks 3′ exonuclease activity, and an isolated thermostable ligase, with amounts such that when the DNA molecules are added in the presence of a suitable buffer solution and dNTPs and incubated under isothermal conditions, the two or more DNA molecules are assembled in a concerted reaction.
Single-vessel one-step thermocycled overlap-based DNA joining with 3′-to-5′ exonuclease
An in vitro method of joining a set of two or more ds or ss DNA molecules in a single vessel by contacting the DNA molecules with an isolated non-thermostable 3′ to 5′ exonuclease active in the presence of dNTPs, a crowding agent, an isolated heat-activated DNA polymerase, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer under conditions effective for joining the DNA molecules to form a first assembled dsDNA molecule in a one-step thermocycled reaction.
One-step thermocycled overlap-based DNA joining kit
A kit for a one-step in vitro reaction to join a set of two or more ds or ss DNA molecules with adjacent molecules containing overlapping sequences at their termini, comprising in a single vessel an isolated non-thermostable 3′ to 5′ exonuclease active in the presence of dNTPs, a crowding agent, an isolated heat-activated DNA polymerase, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer, with amounts such that when the DNA molecules are added and incubated under thermocycled conditions, the DNA molecules are assembled in a concerted reaction.
Representationally dividing and combinatorially assembling whole nucleic acid variants for property modification
A method of modifying the properties of a whole nucleic acid molecule comprising representationally dividing the nucleic acid sequence into portions along its length, identifying sequences of partial nucleic acid molecules, providing, for at least a specified number of the partial nucleic acid molecules, a multiplicity of variants, combinatorially assembling in vitro the variants along with any partial nucleic acid molecules which are not varied where the partial nucleic acid molecules or variants thereof contain overlapping sequences at their termini, and expressing the variants to determine modified properties.
Whole nucleic acid variant modification using degenerate codons, transcription/translation control sequences, or domain/motif regions with metabolic pathway constraint
A method of modifying the properties of a whole nucleic acid molecule by combinatorially assembling in vitro variants of partial nucleic acid molecules with overlapping termini and expressing the variants to determine modified properties, wherein the variants provide degenerate forms of the codon for one or more amino acids encoded by the partial nucleic acid molecules, or provide a multiplicity of nucleic acid control sequences affecting transcription or translation, or provide a multiplicity of regions encoding domains or motifs of peptides or proteins encoded by the whole nucleic acid molecule, or wherein peptides or proteins encoded by the partial nucleic acid molecules function together in a metabolic pathway.
The independent claims collectively define overlap-based assembly of two or more DNA molecules in a single vessel using specified exonuclease/polymerase/ligase systems and crowding with dNTPs and buffer, in either isothermal or thermocycled formats, and define methods for modifying whole nucleic acid properties through representational division, multiplicity-of-variants creation, overlap-based in vitro combinatorial assembly, and expressing the resulting whole-molecule variants to determine modified properties.
Stated Advantages
Defined, reproducible conditions for joining multiple DNA molecules.
Reduced reliance on restriction sites.
Scalable assembly to many fragments.
Automation and high-throughput screening possible.
Ability to handle unstable or recalcitrant DNA.
In vitro repair reduces need for host transformation.
Documented Applications
Codon optimization.
Gene optimization.
Motif/domain control optimization.
Metabolic/pathway optimization.
Minimal genome assembly and large genome assembly, including synthetic Mycoplasma genitalium genome.
Acetate utilization pathway optimization in E. coli.
Mouse mitochondrial genome assembly.
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