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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.:
SGI-DNA, Inc., San Diego, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1632627
Patent Number(s):
10577629
Application Number:
15/385,537
Assignee:
SGI-DNA, Inc. (San Diego, CA)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12Y - ENZYMES
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
DOE Contract Number:  
FC02-02ER63453
Resource Type:
Patent
Resource Relation:
Patent File Date: 12/20/2016
Country of Publication:
United States
Language:
English

Citation Formats

Young, Lei, Smith, Hamilton O., and Gibson, Daniel Glenn. In vitro recombination method. United States: N. p., 2020. 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/1632627.
@article{osti_1632627,
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 = {2020},
month = {3}
}