Abstract
The gene capture technique is a powerful tool that allows the cloning of large DNA regions (up to 80 kb), such as entire genomic islands, without using restriction enzymes or DNA amplification. This technique takes advantage of the high recombinant capacity of the yeast. A “capture” vector containing both ends of the target DNA region must first be constructed. The target region is then captured by co-transformation and recombination in yeast between the “capture” vector and appropriate genomic DNA. The selected recombinant plasmid can be verified by sequencing and transferred in the bacteria for multiple applications.
This chapter describes a protocol specifically adapted for Pseudomonas aeruginosa genomic DNA capture.
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Acknowledgements
We gratefully thank Stephen Lory for providing the plasmids pLLX13, pLLX8 and the CRY1-2 S. cerevisiae strain.
The work in Romé Voulhoux’s lab is supported by the “3D-Pilus” young researcher ANR grant (ANR-JC 07-183230), the “Pathomics” Era-net PATHO grant (ANR-08-PATH-004-01), and Vaincre La Mucoviscidose.
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Ball, G., Filloux, A., Voulhoux, R. (2014). A Method to Capture Large DNA Fragments from Genomic DNA. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_38
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DOI: https://doi.org/10.1007/978-1-4939-0473-0_38
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