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-10626429-B2
Publication Date
2020-04-21
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 concerns an in vitro method of joining a set of two or more double-stranded or single-stranded DNA molecules in which adjacent DNA molecules to be joined contain overlapping sequences at their termini. The method performs contacting of the DNA molecules in a single vessel to form a first assembled dsDNA molecule in a concerted reaction under isothermal conditions.
The method uses, in a mixture, an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity, a crowding agent selected from polyethylene glycol and dextran, an isolated thermostable DNA polymerase, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer. The components are provided in amounts effective for joining the two or more DNA molecules in the single vessel.
The dependent claims further characterize reaction conditions suitable for digesting unpaired, non-homologous single-stranded DNAs after the joining reaction. The method can include DNA molecules having at one terminus a non-homologous sequence relative to the DNA molecules of interest, with the non-homologous sequences including PCR primer binding regions and/or regions of homology to vector sequences and/or recognition sites for one or more restriction enzymes.
Claims Coverage
The provided claims cover one independent claim for an in vitro single-vessel isothermal DNA joining process for DNA molecules with overlapping termini, using a defined combination of enzymes, a crowding agent, dNTPs, and buffer. Dependent claims refine enzyme and crowding-agent choices and extend the functionality to digest unpaired non-homologous ssDNA and define examples of non-homologous terminus sequences.
Single-vessel isothermal joining with overlapping termini
An in vitro method joining a set of two or more ds or ss DNA molecules in which adjacent DNA molecules have overlapping sequences at their termini by contacting the DNA molecules in a single vessel under isothermal conditions to form a first assembled dsDNA molecule in a concerted reaction.
Non-thermostable 5′ to 3′ exonuclease lacking 3′ activity with crowding agent
A mixture comprising an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity and a crowding agent selected from polyethylene glycol and dextran, used together under the isothermal conditions for joining.
Thermostable polymerase and thermostable ligase with dNTPs and buffer
A mixture further comprising an isolated thermostable DNA polymerase, an isolated thermostable ligase, a mixture of dNTPs, and a suitable buffer, wherein the components are provided in amounts effective for joining under isothermal conditions in the single vessel.
Post-joining conditions suitable for digesting unpaired non-homologous ssDNA
Reaction conditions suitable for digesting unpaired, non-homologous single-stranded DNAs after the joining reaction.
Non-homologous terminus sequences relative to DNA molecules of interest
At least some of the DNA molecules to be joined have, at one terminus, a non-homologous sequence relative to the DNA molecules of interest, in the context of the digestion-enabled conditions after joining.
Non-homologous sequences include PCR primer binding regions, vector homology, and restriction recognition sites
The non-homologous sequences include PCR primer binding regions and/or regions of homology to vector sequences and/or recognition sites for one or more restriction enzymes.
The claims center on single-vessel, isothermal in vitro joining of DNA molecules with overlapping termini using a defined mixture that includes a non-thermostable 5′ to 3′ exonuclease lacking 3′ activity, PEG or dextran crowding, a thermostable DNA polymerase, and a thermostable ligase with dNTPs and buffer. Dependent coverage further includes digestion of unpaired non-homologous ssDNA and examples of non-homologous terminus sequence content.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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