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Nucleic Acids Research, 2003, Vol. 31, No. 15 e81
© 2003 Oxford University Press

Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method

Xin-Mei Zhang and Jian-Dong Huang*

Department of Biochemistry, The University of Hong Kong, 3/F Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong SAR, China

*To whom correspondence should be addressed. Tel: +852 2819 2810; Fax: +852 2855 1254; Email: jdhuang{at}hkucc.hku.hk

Recombinogenic engineering or recombineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. We report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (BACs) to build larger, unified BACs. In order to test the feasibility of using recombineering to combine two large DNA fragments (>20 kb), we constructed a unified BAC containing the full-length tyrosinase-related protein-1 (Tyrp-1) gene from two library-derived BACs, one containing the 5' regulatory elements and the other containing the 3' coding exons. This was achieved using a two-step homologous recombination method enabled by the bacteriophage {lambda} Red proteins. In the first step, retrieval, a large DNA fragment (~22 kb) was retrieved from one of the original BACs. In the second step, recombination, the retrieved DNA fragment was inserted into the second original BAC to form the unified BAC containing all the desired Tyrp-1 sequence. To further demonstrate the general applicability of our approach, an additional DNA fragment (~20 kb) was inserted into the unified BAC downstream of the coding region. This method should prove very useful for enabling BAC manipulation in a variety of scenarios.


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