IN VITRO RECOMBINATION METHOD
First Claim
1. A kit for in vitro joining a plurality of dsDNA molecules, comprising, in separate containers,(a) a mixture of the isolated proteins(i) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing,(ii) a non strand-displacing DNA polymerase, and(iii) a ligase,wherein the ratios of activities of (i), (ii) and (iii) are effective, when in the presence of a non-processive 5′
- exonuclease, to achieve in vitro joining of dsDNA molecules, and(b) an isolated non-processive 5′
exonuclease.
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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.
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Citations
3 Claims
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1. A kit for in vitro joining a plurality of dsDNA molecules, comprising, in separate containers,
(a) a mixture of the isolated proteins (i) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing, (ii) a non strand-displacing DNA polymerase, and (iii) a ligase, wherein the ratios of activities of (i), (ii) and (iii) are effective, when in the presence of a non-processive 5′ - exonuclease, to achieve in vitro joining of dsDNA molecules, and
(b) an isolated non-processive 5′
exonuclease.
- exonuclease, to achieve in vitro joining of dsDNA molecules, and
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2. The kit of paragraph 1, wherein
(a) is a mixture of (i) as the SSB, the phage T7 gene 2.5 product, the E. coli recA protein, RedB of lambda phage, or RecT of Rac prophage; -
(ii) as the DNA polymerase, the T7 gene 5 product, T4 polymerase, or E. coli pol; and
/or(iii) as a ligase, the phage T7 gene 1.3 product, phage T4DNA ligase, or E. coli DNA ligase; and
/or(b) is, as the 5′
exonuclease, the phage T7 gene 6 product, RedA of lambda phage, or RecE of Rac prophage.
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3. A composition comprising
(a) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing, (b) a non strand-displacing DNA polymerase, (c) a ligase, and (d) an isolated-non-processive 5′ - exonuclease,
wherein the ratios of activities of (a), (b), (c) and (d) are effective to achieve in vitro joining of dsDNA molecules.
- exonuclease,
Specification