Molecular Biotechnology

, Volume 31, Issue 3, pp 181–186 | Cite as

A general method to modify BACs to generate large recombinant DNA fragments

  • Wei Shen
  • Yue Huang
  • Yi Tang
  • De-Pei Liu
  • Chih-Chuan Liang
Research

Abstract

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA frament containing the inverted human α-globin genes (ϑ, α1, α2, and ζ) from BAC191K2 and the locus control region (LCR) of human β-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

Index Entries

Bacterial artificial chromosomes recombineering rare-cut restriction endonuclease 

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Wei Shen
  • Yue Huang
  • Yi Tang
  • De-Pei Liu
    • 1
  • Chih-Chuan Liang
  1. 1.National Laboratory of Medical Molecular Biology, Institute of Basic Medical SciencesChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina

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