Method for in vitro recombination
The present invention relates, e.g., to an 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 (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.
- Research Organization:
- Synthetic Genomics, Inc., San Diego, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-02ER63453
- Assignee:
- Synthetic Genomics, Inc. (San Diego, CA)
- Patent Number(s):
- 7,776,532
- Application Number:
- 11/502,624
- OSTI ID:
- 1531645
- Resource Relation:
- Patent File Date: 2006-08-11
- Country of Publication:
- United States
- Language:
- English
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In vitro recombination method
In vitro recombination method