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Nucleic Acids Research, 2001, Vol. 29, No. 7 e37
© 2001 Oxford University Press

Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli

Margherita Sosio, Elena Bossi and Stefano Donadio*

Biosearch Italia SpA, Via R. Lepetit 34, 21040 Gerenzano, Italy

We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC). The procedure makes use of iterative rounds of homologous recombination in Escherichia coli. Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required. They are transferred first into a temperature-sensitive replicon and then into the BAC of choice. We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli–Streptomyces artificial chromosome (ESAC). Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids.

* To whom correspondence should be addressed. Tel: +39 02 9647 4243; Fax: +39 02 9647 4238; Email: sdonadio{at}biosearch.it


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