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Homologous recombination-based DNA cloning methods and compositions

a dna cloning and homologous technology, applied in the field of molecular cloning methods and compositions, can solve the problems of limiting the user to particular vectors and host cells, cumbersome and time-consuming conventional cloning methods, and low cloning efficiency

Inactive Publication Date: 2010-03-11
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method, composition, kit, and system for cloning a donor DNA molecule into an acceptor vector at a predetermined location. This involves preparing an extended donor DNA molecule by adding specific sequences to its ends, incubating it with an enzyme cocktail, and transforming a cell with the resulting intermediate product. The invention allows for more precise and efficient cloning of donor DNA into an acceptor vector.

Problems solved by technology

The conventional cloning method is cumbersome and time-consuming.
It has relatively low cloning efficiency.
It is also limited by the availability of suitable restriction enzyme recognition sites on the vector and the donor DNA.
Like several other recombination-based methods, the method depends on specific sequences within the acceptor vector and the expression of specific phage proteins in the host cell, thus restricts the user to particular vectors and host cells.
While the In-Fusion™ system does allow rapid directional cloning of PCR product without the restrictions of vectors and host cells, it depends on the proprietary In-Fusion™ enzyme, thus restricts the user to the In-Fusion™ PCR Cloning Kits or similar systems sold by Clontech or its affiliates.

Method used

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  • Homologous recombination-based DNA cloning methods and compositions

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Embodiment Construction

[0033]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0034]As used herein, “sequence” means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

[0035]As used herein, the term “nucleotide sequence”, “nucleic acid” or “polynucleotide” refers to the arrangement of either deoxyribonucleotide or ribonucleotide residues in a polymer in either single- or double-s...

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Abstract

Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is entitled to and claims the benefit of the priority pursuant to 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61 / 095,877, filed Sep. 10, 2008, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]Embodiments of the present invention relate to methods and compositions for molecular cloning, particularly, cloning of a donor DNA into an acceptor vector at a predetermined location.[0004]2. Background of the Invention[0005]In molecular biology research and biotechnology industry, there is a constant need of cloning a desired DNA molecule into a vector, preferably at a predetermined location. A conventional method for cloning a donor DNA into an acceptor vector at a predetermined location, such as a plasmid, usually involves six major steps: (i) digesting the acceptor vector DNA with one or two restriction endonucleases and p...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/64C12N9/16
CPCC12N15/66
Inventor LIU, WEIQIANGYANG, PINGWANG, TAOWANG, ZHUYINGCHEN, WENZHUZHANG, FANG LIANG
Owner NANJING GENSCRIPT BIOTECH CO LTD
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