Abstract
SLiCE (Seamless Ligation Cloning Extract) is a novel cloning method that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (15–52 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from bacterial artificial chromosomes or other sources. SLiCE is highly cost-effective and demonstrates the versatility as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. We established a DH10B-derived E. coli strain expressing an optimized λ prophage Red recombination system, termed PPY, which facilitates SLiCE with very high efficiencies.
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References
Smith HO, Wilcox KW (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 51:379–391
Danna K, Nathans D (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc Natl Acad Sci U S A 68:2913–2917
Cohen SN, Chang AC, Boyer HW et al (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 70:3240–3244
Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40(8):e55
Little JW (1967) An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. J Biol Chem 242(4):679–686
Radding CM, Carter DM (1971) The role of exonuclease and beta protein of phage lambda in genetic recombination. 3. Binding to deoxyribonucleic acid. J Biol Chem 246(8):2513–2518
Carter DM, Radding CM (1971) The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J Biol Chem 246(8):2502–2512
Lovett ST, Hurley RL, Sutera VA et al (2002) Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways. Genetics 160:851–859
Dutra BE, Sutera VA, Lovett ST (2007) RecA-independent recombination is efficient but limited by exonucleases. Proc Natl Acad Sci U S A 104:216–221
Kuzminov A (2002) Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol Mol Biol Rev 63:751–813
Acknowledgement
This work was supported by National Institute of Health [1R01CA76329 to W.E., 1R01CA93484 to W.E].
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Zhang, Y., Werling, U., Edelmann, W. (2014). Seamless Ligation Cloning Extract (SLiCE) Cloning Method. In: Valla, S., Lale, R. (eds) DNA Cloning and Assembly Methods. Methods in Molecular Biology, vol 1116. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-764-8_16
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DOI: https://doi.org/10.1007/978-1-62703-764-8_16
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