In vitro recombination method
Inventors
Young, Lei • Smith, Hamilton O. • Gibson, Daniel Glenn
Assignees
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Publication Number
US-11542529-B2
Publication Date
2023-01-03
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 relates to an in vitro DNA joining/recombination method that uses isolated proteins to join at least two double strand DNA molecules of interest. The dsDNA molecules share a region of sequence identity of at least 15 base pairs at a terminal end on each DNA molecule, and the method provides a 3′ single-stranded overhang in each molecule by contacting the dsDNA with a purified 5′ exonuclease without use of a restriction enzyme.
The two 3′ single-stranded overhangs anneal to form a gapped molecule, and the gaps are filled in by a purified DNA polymerase. Nicks are then sealed by a purified ligase to form a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained.
The described approach supports joining multiple DNA fragments in a predetermined order/orientation and restriction enzyme-free assembly using shared terminal identity regions. The document further reports formation of circular DNA products or vector assembly.
Claims Coverage
The partial document includes one independent claim, with dependent claims refining the same core in vitro restriction enzyme-free joining workflow. The independent claim specifies five main inventive features.
Purified exonuclease-generated 3′ single-stranded overhangs without restriction enzyme
A purified 5′ exonuclease generates a 3′ single-stranded overhang in each dsDNA molecule at a terminal end, without the use of a restriction enzyme.
Annealing into a gapped intermediate from shared terminal identity
The dsDNA molecules anneal through the two single-stranded overhangs to form a gapped molecule.
Polymerase gap filling to create substantially intact double stranded DNA
A purified DNA polymerase fills the gaps of the gapped molecule.
Ligase nick sealing with retention of a single copy of the sequence identity region
A purified ligase seals nicks to join the molecules and form a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained.
Coordinated reaction sequencing without active termination between steps
None of the enzymatic reactions is actively terminated prior to beginning another of the reactions.
Overall, the claim coverage focuses on a coordinated, restriction enzyme-free in vitro joining process using isolated proteins: purified 5′ exonuclease generates 3′ overhangs, overhangs anneal to form gapped intermediates, purified DNA polymerase fills gaps, and purified ligase seals nicks, while retaining a single copy of the shared terminal identity region and maintaining an enzymatic sequencing condition regarding reaction termination.
Stated Advantages
Enables joining that does not require a restriction enzyme.
Retains a single copy of the region of sequence identity in the joined double stranded DNA molecule.
Allows joining multiple DNA fragments in a predetermined order/orientation.
Supports assembly into circular products or vectors.
Documented Applications
Assembly of multiple DNA fragments into a joined product.
Circular DNA formation.
Vector or circular DNA assembly context.
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