Int J Biochem Mol Biol 2011;2(3):199-206

Original Article
A multi-step strategy for BAC recombineering of large DNA fragments

Yuanjun Zhao, Shuwen Wang, Jiyue Zhu

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033

Received April 18, 2011; accepted May 15, 2011; Epub May 20, 2010; Published August 30, 2011

Abstract: Recombineering techniques have been developed to modify bacterial artificial chromosomes (BACs) via bacterial homologous
recombination systems, simplifying the molecular manipulations of large DNA constructs. However, precise modifications of a DNA fragment
larger than 2-3 kb by recombineering remain a difficult task, due to technical limitations in PCR amplification and purification of large DNA
fragments. Here, we describe a new recombineering strategy for the replacement of large DNA fragments using the commonly utilized lambda
phage/Red recombination host system. This approach involved the introduction of rare restriction enzyme sites and positive selection markers
into the ends of a large DNA fragment, followed by its release from the donor BAC construct and integration into an acceptor BAC. We have
successfully employed this method to precisely swap a number of large DNA fragments ranging from 6 to 40 kb between two BAC constructs.
Our results demonstrated that this new strategy was highly effective in the manipulations of large genomic DNA fragments and therefore
should advance the conventional BAC recombineering technology to the next level. (IJBMB1104005).

Keywords: Bacterial artificial chromosome, recombineering, homologous recombination, chimeric BACs, large DNA fragments

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