In vitro recombination method

Young, Lei; Smith, Hamilton O.; Gibson, Daniel Glenn

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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:: Young, Lei; Smith, Hamilton O.; Gibson, Daniel Glenn

Issue Date:: 2023-01-03

Research Org.:: Codex DNA, Inc., San Diego, CA (United States)

Sponsoring Org.:: USDOE

OSTI Identifier:: 1986987

Patent Number(s):: 11542529

Application Number:: 16/799,424

Assignee:: Codex DNA, Inc. (San Diego, CA)

DOE Contract Number:: FG02-02ER63453

Resource Type:: Patent

Resource Relation:: Patent File Date: 02/24/2020

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., 2023. 
        Web.

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                    Young, Lei, Smith, Hamilton O., & Gibson, Daniel Glenn. In vitro recombination method.  United States.

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                    Young, Lei, Smith, Hamilton O., and Gibson, Daniel Glenn. Tue .  
        "In vitro recombination method".  United States.  https://www.osti.gov/servlets/purl/1986987.

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@article{osti_1986987,

  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         = {2023},

  month        = {1}

}

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Works referenced in this record:

TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing
journal, June 2002

Nelson, John R.; Cai, Yuyang Christine; Giesler, Theresa L.
BioTechniques, Vol. 32, Issue 6S
https://doi.org/10.2144/jun0208

Method for Preparing Water-Insoluble Alpha-1, 4-Glucans
patent-application, May 2002

Quanz, Martin; Provart, Nicholas; Banasiak, Ronald
US Patent Application 09/740824; 20020052029
URL: https://image-ppubs.uspto.gov/dirsearch-public/print/downloadPdf/20020052029