In vitro recombination method
Inventors
Young, Lei • Smith, Hamilton O. • Gibson, Daniel Glenn
Assignees
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Publication Number
US-10577629-B2
Publication Date
2020-03-03
Expiration Date
Abstract
The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.
Core Innovation
The invention provides an in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest that share a region of sequence identity at a terminal end on each DNA molecule. The method contacts the first and second dsDNA molecules with a purified 5′ exonuclease, a purified DNA polymerase, and a purified ligase, without the use of a restriction enzyme. A 3′ single-stranded overhang is generated in each molecule by the exonuclease, and the overhangs anneal to form a gapped molecule.
After annealing, the gaps are filled in by the purified DNA polymerase and nicks are sealed by the purified ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule. The method retains a single copy of the region of sequence identity in the resulting substantially intact double stranded DNA molecule. The reactions are controlled such that none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.
The disclosed approach enables joining multiple dsDNA fragments in predetermined order and orientation using overlap or region of homology, while avoiding restriction enzymes. It is described as an in vitro, isolated-protein assembly that retains a single copy of a terminal region of sequence identity and supports controlled, reproducible assembly under defined conditions. The disclosure also highlights joining overlaps shorter than about 150 bp and includes example results demonstrating formation of an expected joined DNA product and circular DNA recombination.
Claims Coverage
Independent claim clm-00001 covers the core in vitro isolated-protein joining workflow using a purified 5′ exonuclease, a purified DNA polymerase, and a purified ligase to join terminally homologous dsDNA molecules while retaining a single copy of the region of sequence identity.
Isolated-protein in vitro joining of terminally homologous dsDNA
An in vitro method, using isolated proteins, for joining two dsDNA molecules of interest that share a region of sequence identity at a terminal end on each DNA molecule.
Purified 5′ exonuclease generates 3′ overhangs without restriction enzyme
Contacting the two dsDNA molecules with a purified 5′ exonuclease to generate a 3′ single-stranded overhang in each molecule without the use of a restriction enzyme.
Annealing to form a gapped molecule followed by polymerase fill-in
The two single-stranded overhangs anneal to form a gapped molecule, and the gaps are filled in by the purified DNA polymerase.
Ligase seals nicks to form substantially intact DNA retaining a single copy
Nicks are sealed by the purified ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule in which a single copy of the region of sequence identity is retained.
No active termination prior to beginning another reaction
None of the enzymatic reactions is actively terminated prior to beginning another of the reactions.
Across the independent claim, the coverage centers on an isolated-protein, in vitro dsDNA joining scheme driven by purified 5′ exonuclease-generated 3′ overhangs, followed by gapped annealing, polymerase gap filling, and ligase nick sealing, with retention of a single copy of the terminal region of sequence identity.
Stated Advantages
Controlled and reproducible assembly under defined conditions.
Joining multiple dsDNA fragments in predetermined order and orientation.
Restriction enzyme independence.
Joining overlaps shorter than about 150 bp.
Documented Applications
Joining DNA fragments to form an expected joined product of about 6.3 kb, as demonstrated by example results.
Circular DNA recombination, including an example of circular DNA formation.
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