Methods, compositions and kits for a one-step DNA cloning system
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Publication Number
US-10253321-B2
Publication Date
2019-04-09
Expiration Date
Abstract
Methods and kits for joining three or more polynucleotides to form a product polynucleotide are provided. Such a method includes forming a reaction mixture comprising (i) a vector fragment whose ends have four base overhangs resulting from cleavage of a vector with a first type IIs enzyme, (ii) a first insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by the second type IIs enzyme; and (iii) a second insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by the second type IIs enzyme, and (iv) a ligase. The four base overhangs of the vector ligate with the four base overhangs of the first and second inserts and the three base overhangs of the first and second inserts ligate with each other or three base overhangs of a spacer nucleic acid resulting from cleavage with the second type IIs enzyme to form a product polynucleotide in which the first and second insert nucleic acids are joined to the vector fragment.
Core Innovation
The invention provides a method of forming a product polynucleotide by forming a reaction mixture including a vector fragment and multiple insert nucleic acids, and then ligating them by a single reaction of joining multiple polynucleotides. The vector fragment has four base overhangs resulting from cleavage of the vector with a first type IIs enzyme, and each insert nucleic acid has a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by a second type IIs enzyme.
The four base overhangs of the vector ligate with the four base overhangs of the first and second inserts, while the three base overhangs of the first and second inserts ligate with each other or with three base overhangs of a spacer nucleic acid resulting from cleavage with the second type IIs enzyme. This arrangement forms a product polynucleotide in which the first and second insert nucleic acids are joined to the vector fragment by a single reaction. The system is described as assembling three or more polynucleotides in a single reaction using type IIs enzyme-generated overhangs.
The document describes directed fusion/ORF junction outcomes produced by ligation of three-base overhangs, including initiator and stop arrangements and codon linker outcomes such as glycine/alanine codons. It further describes constructs including marker vectors with selectable and counter-selectable marker vectors, and kits containing enzyme, buffer, and instructions for the one-step multi-fragment assembly. The assembly is illustrated with an antibody example (Herceptin) and is described as forming scarless junctions with expression validated.
Claims Coverage
The independent claim recites a single-reaction method forming a product polynucleotide by joining a vector fragment, a first insert nucleic acid, and a second insert nucleic acid using type IIs enzyme-generated overhangs, with four-base overhang compatibility and three-base overhang junction control. The inventive features include the specific overhang structure and ligation logic that enables the first and second insert nucleic acids to be joined to the vector fragment in one joining reaction, including an optional spacer nucleic acid path for the three-base overhangs.
Single reaction joining of vector and multiple inserts using distinct overhang classes
forming a reaction mixture comprising (i) a vector fragment whose ends have four base overhangs resulting from cleavage of a vector with a first type IIs enzyme, (ii) a first insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by a second type IIs enzyme; and (iii) a second insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by the second type IIs enzyme, and (iv) a ligase, wherein the four base overhangs of the vector ligate with the four base overhangs of the first and second inserts and the three base overhangs of the first and second inserts ligate with each other or three base overhangs of a spacer nucleic acid resulting from cleavage with the second type IIs enzyme to form a product polynucleotide in which the first and second insert nucleic acids are joined to the vector fragment by a single reaction of joining multiple polynucleotides.
Four base overhang generation from the first type IIs enzyme for vector-to-inserts ligation
the vector fragment ends have four base overhangs resulting from cleavage of a vector with a first type IIs enzyme, and the first and second insert nucleic acids each have a four base overhang at one end resulting from cleavage by the first type IIs enzyme, so that the four base overhangs of the vector ligate with the four base overhangs of the first and second inserts.
Three base overhang junction control between inserts or via a spacer nucleic acid
the first and second insert nucleic acids each have a three base overhang at the other end resulting from cleavage by a second type IIs enzyme, wherein the three base overhangs of the first and second inserts ligate with each other or ligate with three base overhangs of a spacer nucleic acid resulting from cleavage with the second type IIs enzyme to form the product polynucleotide.
Overhang formation using specific type IIs enzymes
the first type IIs enzyme is BsaI and the second type IIs enzyme is SapI.
Spacer nucleic acid with three base overhangs from second type IIs cleavage
a spacer nucleic acid with three-base overhangs at each end from cleavage by a second type IIs enzyme, and ligates these overhangs with three-base overhangs of first and second insert nucleic acids to form a product polynucleotide joined to a vector fragment.
Vector fragment generation from counter-selectable marker vector or linear vector
a vector fragment is generated in a reaction mixture by cleavage of either a vector with a counter-selectable marker or a linear vector.
Counter-selectable marker selection for vector preparation
selecting the counter-selectable marker from the group including sacB, rpsL, tetAR, pheS, thyA, lacY, gata-1, ccdB, kid, barnase.
Glycine or alanine codon formation from three-base overhang ligation in a fusion protein
ligating three-base overhangs of a first and second insert nucleic acid to generate a glycine or alanine codon that encodes a glycine or alanine between two polypeptides in a fusion protein.
Across the independent claim and its dependents, coverage centers on forming a product polynucleotide by a single reaction that uses type IIs enzyme-generated four-base overhangs for vector-to-insert ligation and three-base overhangs for insert-to-insert or insert-to-spacer junction formation. The dependents further specify enzyme identities, the optional spacer nucleic acid, vector fragment generation via counter-selectable marker vectors or linear vectors, a defined set of counter-selectable markers, and coding outcomes from three-base overhang ligation such as glycine or alanine codon placement for fusion proteins.
Stated Advantages
High correct-configuration efficiency is described for the illustrated assembly.
Scarless junctions are described.
Expression is validated for the antibody example (Herceptin).
Documented Applications
Assembly of an antibody (Herceptin) using the one-step multi-fragment cloning system, with expression validated.
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