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Harnessing Homologous Recombination in Vitro to Generate Recombinant DNA Via SLIC

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Harnessing Homologous Recombination in Vitro to Generate Recombinant DNA Via SLIC ARTICLES Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC methods Mamie Z Li & Stephen J Elledge We describe a new cloning method, sequence and ligation– The third, MAGIC, is an in vivo method that relies upon homo- .com/nature e independent cloning (SLIC), which allows the assembly of logous recombination and bacterial mating15.Thesemethodsoffer multiple DNA fragments in a single reaction using in vitro a uniform and seamless transfer of genes from one expression .natur homologous recombination and single-strand annealing. context to another, thereby allowing different clones to be treated w SLIC mimics in vivo homologous recombination by relying identically. These methods, however, lack important features. First, on exonuclease-generated ssDNA overhangs in insert and they do not generally facilitate initial assembly of the gene of vector fragments, and the assembly of these fragments by interest into the origin plasmid. The exception, Gateway, requires http://ww recombination in vitro. SLIC inserts can also be prepared by the use of expensive enzymes for initial cloning and requires incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and specific long sequences on each primer that contain recombination oup r G reproducible assembly of recombinant DNA with as many as sites. Second, these methods are useful only for cloning into specific 5 and 10 fragments simultaneously. SLIC circumvents the vectors containing defined sequences. If a cloning reaction requires sequence requirements of traditional methods and functions a specialty assembly, that is, replacing a fragment in an existing lishing much more efficiently at very low DNA concentrations when plasmid, perhaps within a gene, these methods cannot be used. b combined with RecA to catalyze homologous recombination. Finally, these methods generally allow only the combination of two Pu This flexibility allows much greater versatility in the generation fragments in a single experiment. of recombinant DNA for the purposes of synthetic biology. Homologous recombination has important advantages over site- Nature specific recombination in that it does not require specific 7 The assembly of recombinant DNA by restriction enzyme cutting sequences. Two types of homologous recombination exist in th 16,17 200 and re-ligation was a crowning achievement of biology in the 20 Escherichia coli , RecA-mediated recombination and a RecA- 1–4 © century . Many variations on this theme have emerged that allow independent pathway called single-strand annealing. We have greater precision to be achieved with respect to sequence alterations addressed limitations of present systems by the development of a and sites of junctions of recombinant molecules. Two methods that new in vitro homologous recombination method called SLIC. We made critical improvements are site-directed mutagenesis5 and show that homologous recombination intermediates, such as large PCR6,7. Site-directed mutagenesis allows alteration of specific gapped molecules assembled in vitro by RecA or single-strand sequences to allow structure-function studies of molecules. PCR annealing, efficiently transform E. coli, removing the sequence has made several contributions including the ability to select a constraints inherent in other methods. This system circumvents precise sequence from low concentrations of DNA and to place many problems associated with conventional cloning methods, specific sequences at fragment ends to allow conventional assembly providing a multifaceted approach for the efficient generation of with other fragments. PCR has also been used to introduce changes recombinant DNA. into gene sequences8. Presently the DNA sequence and coding capacities of entire RESULTS organisms are being determined. This presents the opportunity to In vitro homologous recombination with and without RecA manipulate and analyze large sets of genes for genetic and bio- Homologous recombination in vivo depends upon a double- chemical properties. Furthermore, a new field, synthetic biology, is stranded break, generation of ssDNA by exonucleases, homology emerging, which uses complex combinations of genetic elements to searching by recombinases, annealing of homologous stretches, and design circuits with new properties. These endeavors require the repair of overhangs and gaps by enzymes that include resolvases, development of new cloning technologies. Three recombination- nucleases and polymerases. We reasoned it might be possible to based cloning methods have emerged for accomplishing parallel generate recombination intermediates in vitro and introduce these processing of large gene sets. Two use in vitro site-specific recom- into cells to allow the cell endogenous repair machinery to finish bination, the Univector Plasmid-fusion System and Gateway9–14. the repair to generate recombinant DNA (Fig. 1a). To generate Howard Hughes Medical Institute, Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. Correspondence should be addressed to. S.J.E. ([email protected]). RECEIVED 12 SEPTEMBER 2006; ACCEPTED 4 JANUARY 2007; PUBLISHED ONLINE 11 FEBRUARY 2007; DOI:10.1038/NMETH1010 NATURE METHODS | VOL.4 NO.3 | MARCH 2007 | 251 ARTICLES a Figure 1 | In vitro recombination of MAGIC vectors mediated by RecA. Gene (a) A schematic for production of recombinant DNA through in vitro PCR homologous recombination and single-strand annealing. (b) XmnIandEcoRI Gene restriction maps of MAGIC plasmids pML410-pheS, pSM2c-Rb and recombinant T4 DNA polymerase (exo) 5′ plasmid. (c)AnXmnI-EcoRI restriction analysis of the recombinants derived Gene 5′ from SLIC reaction. Plasmid DNA from ten independent carbenicillin-resistant + ′ 5′ 5 Vector colonies (lanes 1–10) were digested with XmnI-EcoRI. The digestion products were separated on a 0.8% agarose gel and visualized after ethidium bromide With or without RecA staining. Recombinants contain one EcoRI site from the insert and three XmnI sites from the vector (4.2, 3.1, 1.5 and 1.4 kb). Gene Recombination intermediate Transformation twofold reduction in background. Transformation with vector alone yielded very few transformants, whereas incubation of methods Recombinant plasmid the vector with equimolar insert fragments and RecA produced 400–600-fold increase over background. All 20 clones analyzed had b Xmnl Xmnl the correct restriction map (data not shown). We observed an Xmnl Xmnl 1.5 kb 1.5 kb .com/nature increase in formation of recombinants over background with e addition of an insert in the absence of RecA, indicating that pML410-pheS 3.1 kb pSM2c-Rb Recombinant 3.1 kb 5.5 kb 4.2 kb single-strand annealing could produce recombinants under these .natur 10.1 kb 7.2 kb 10.2 kb w conditions. By varying the length of overlap, we found that 30-bp pheS Xmnl ShRNA ShRNA Xmnl homology gave the greatest stimulation of recombination (Table 1). 1.4 kb To examine how this method might apply to different vector and EcoRI I-SceI I-SceI I-SceI I-SceI EcoRI http://ww insert systems, we chose a vector-insert combination previously c M12345678910 shown to efficiently recombine in vivo via MAGIC cloning. MAGIC oup donor vectors have greater than 60 bp homology with recipient r 4.2 kb G 3.1 kb vectors on each end and generate inserts by cleavage with I-SceIof 1.5 kb 1.4 kb both donor and recipient plasmids. The recipient used was a lentivector and the donor fragment was a short hairpin RNA lishing b cassette from a short hairpin RNA library. We incubated prepared Pu overhangs for homology searching, we used exonucleases to chew fragments with and without RecA, electroporated into E. coli and back one strand to reveal ssDNA overhangs. We tested both 3¢ and selected for carbenicillin resistance. Without RecA, vector alone 5¢ exonucleases including T7 exonuclease, lambda exonuclease and gave 1.3 transformants per nanogram of vector, whereas vector plus Nature 18 T4 DNA polymerase . We chose T4 DNA polymerase, which insert gave 51. With RecA, vector alone gave 8 transformants per 7 produces 5¢ overhangs, because it gave the best and most repro- nanogram of vector whereas vector plus insert gave 3,900 transfor- 200 ducible results (Supplementary Table 1 online), and had the ability mants, a 500-fold stimulation of recombination, similar to what we © to terminate excision by addition of a single dNTP. observed in vivo. Ten of ten clones yielded restriction fragments For the cloning reaction we generated the vector by cleavage with consistent with the predicted restriction map (Fig. 1b,c). Addi- a restriction enzyme and generated the insert by PCR. We treated tionally, the isolation of 3,900 transformants per nanogram of both the vector and the insert with T4 DNA polymerase in the vector means libraries can be transferred by this method in vitro absence of dNTPs to generate overhangs, then incubated vector and without losing complexity. insert with and without RecA protein and ATP to promote The requirement of RecA for efficient recombination suggested recombination, and transformed the products into E. coli. Trans- that at the DNA concentrations used, efficient homology searching formation with vector alone gave some background, which we required enzymatic facilitation. We wondered whether the less traced to a small amount of uncleaved vector. We reduced the efficient single-strand annealing pathway could be made more background in these experiments by placing the negative selection marker pheS A294G between the restriction sites in the vector and 1,200 growing transformants on plates containing chlorophenylalanine. –RecA In most cases however, chlorophenylalanine selection gave only a 1,000 +RecA 800 600 Table 1 | Stimulation of recombination by RecA (c.f.u./ng) 400 20 bp 30 bp 40 bp 50 bp Amount of recombinants 200 a 0 Vector only o2.7 8.1 2.7 o2.7 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Vectora and Skp1 210 440 89 120 Vector concentration (ng/µl) Vectora and RecA 2.7 o2.7 o2.7 o2.7 Figure 2 | The dependency on RecA can be overcome by increasing DNA Vectora and Skp1 and RecA 1,700 4,900 1,200 1,300 concentration.
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