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

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Zhang, Xinmei and Huang, Jian‐Dong (2003) Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method. Nucleic Acids Research, 31 (15). pp. 1-6. ISSN 1362-4962

Abstract

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 λ 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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Additional Information

Online document no: e81

Item type Article
URI https://vuir.vu.edu.au/id/eprint/21760
DOI https://doi.org/10.1093/nar/gng081
Official URL http://nar.oxfordjournals.org/content/31/15/e81
Subjects Current > FOR Classification > 1004 Medical Biotechnology
Current > FOR Classification > 1103 Clinical Sciences
Historical > Faculty/School/Research Centre/Department > School of Sport and Exercise Science
Keywords cloning vector, DNA manipulation, tyrosinase-related protein-1 gene, Tyrp-1 gene, human artificial chromosomes, HACs, gene function, E.coli chromosome, DNA fragments
Citations in Scopus 17 - View on Scopus
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