Abstract
The One-Step Isothermal DNA Assembly method allows for the efficient assembly of DNA constructs using fragments up to several hundred kilobases in as little as 15 min. Applications of this method range from the addition of promoters to expression constructs to the assembly of bacterial genome fragments. The production of circularized DNA using this method also enables the direct transformation of target organisms, bypassing intermediate transformations for plasmid propagation in those species where expression could lead to toxicity and cell death. Variations of the method allow for specific cloning tasks to be performed, as well as the use of microarray slides as a source of DNA. The level of precision and simplicity of this method makes it a valuable tool for most cloning efforts and all levels of proficiency in molecular biology.
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References
Gibson DG, Benders GA et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220
Gibson DG, Glass JI et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56
Gibson DG, Smith HO et al (2010) Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7:901–903
Krishnakumar R, Assad-Garcia N et al (2010) Targeted chromosomal knockouts in Mycoplasma pneumoniae. Appl Environ Microbiol 76:5297–5299
Widmaier DM, Voigt CA (2010) Quantification of the physiochemical constraints on the export of spider silk proteins by salmonella type III secretion. Microb Cell Fact 9:78–90
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Rodrigues, R.T.L., Bayer, T.S. (2013). One-Step Isothermal Assembly of DNA Fragments. In: Polizzi, K., Kontoravdi, C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1073. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-625-2_5
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DOI: https://doi.org/10.1007/978-1-62703-625-2_5
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Publisher Name: Humana Press, Totowa, NJ
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