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Title: In vitro recombination method

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.

Inventors:
; ;
Issue Date:
Research Org.:
Synthetic Genomics, Inc., La Jolla, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1531965
Patent Number(s):
9,534,251
Application Number:
12/750,571
Assignee:
Synthetic Genomics, Inc. (La Jolla, CA)
DOE Contract Number:  
FG02-02ER63453
Resource Type:
Patent
Resource Relation:
Patent File Date: 2010-03-30
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Young, Lei, Smith, Hamilton O., and Gibson, Daniel Glenn. In vitro recombination method. United States: N. p., 2017. Web.
Young, Lei, Smith, Hamilton O., & Gibson, Daniel Glenn. In vitro recombination method. United States.
Young, Lei, Smith, Hamilton O., and Gibson, Daniel Glenn. Tue . "In vitro recombination method". United States. https://www.osti.gov/servlets/purl/1531965.
@article{osti_1531965,
title = {In vitro recombination method},
author = {Young, Lei and Smith, Hamilton O. and Gibson, Daniel Glenn},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2017},
month = {1}
}

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Works referenced in this record:

Process for in vitro creation of recombinant polynucleotide sequences by oriented ligation
patent, January 2006


Method for amplification of nucleic acids in solid media
patent, April 1997


Directional cloning
patent, December 1991


Synthetic genomes
patent-application, November 2007


Stimulation of enzymatic ligation of DNA by high concentrations of nonspecific polymers
patent, April 1986


Method of analysis and manipulation of DNA utilizing mismatch repair systems
patent, December 1999


Methods and kits for recombining nucleic acids
patent, November 2000


Thermostable DNA ligase mutants
patent, June 2003


Evolution of whole cells and organisms by recursive sequence recombination
patent, April 2002


Method for nucleic acid hybridization using single-stranded DNA binding protein
patent, July 1996


    Works referencing / citing this record:

    Efficient and rapid method for assembling and cloning double-stranded DNA fragments
    patent, April 2018