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Nucleic Acids Research Advance Access published online on November 6, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn890
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes

Fuchun Zhou1,2, Qiuhua Li1,3 and Shou-Jiang Gao1,2,3,4,5,6,7,*

1Tumor Virology Program, Greehey Children's Cancer Research Institute, 2Department of Pediatrics, 3Department of Microbiology and Immunology, 4Department of Molecular Medicine, 5Department of Medicine, 6Department of Cancer Therapy and Research Center, The University of Texas Health Science Center, San Antonio, TX 78229, USA and 7Tumor Virology Group, Wuhan Institute of Virology, Chinese Academy of Sciences, Xiao Hong Shan Lu, Wuhan, China

*To whom correspondence should be addressed. Tel: +1 210 562 9030; Fax: +1 210 562 9014; Email: gaos{at}uthscsa.edu

Received May 17, 2008. Revised October 21, 2008. Accepted October 21, 2008.

Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.


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