BAC Library Construction

Abstract

DNA cloning, especially large DNA cloning, is the first step in contemporary complex genome analysis. Cloning technology of high-molecular-weight DNA has been developed mainly using yeast and Escherichia coli as hosts. In the early stages of the Human Genome Project, yeast artificial chromosome (YAC) libraries have been generated and used for construction of a framework of the genome. The YAC cloning system has a great advantage of cloning of very large (>500 kb) DNA, thus facilitating construction of a physical map of the complex genome. The bacterial artificial chromosome (BAC) technologies matured later but proved to have so many advantages that the BAC libraries have been the primary input to contig assembly and the public sector human genome sequencing. BACs are easily purified as plasmid DNAs, have little if any chimerism, and are stable, with a very few interesting exceptions. Both BAC and bacteriophage P1-derived artificial chromosome (PAC) cloning systems have been developed, respectively, using the E. coli F-factor plasmid replication and bacteriophage P1 plasmid origin to maintain largeness (100–250 kb). Genomic DNA is subjected to partial digestion with a restriction endonuclease in order to break DNA into clonable size and size fractionated using pulsed-field gel electrophoresis (PFGE). The size-fractionated DNA is cloned into a BAC vector and transformed into E. coli by electrical shock. The transformants are arrayed into microtiter dishes and high-density replica filters are prepared to facilitate screening of the library. Human genome draft sequences were reported using two different (BAC clone-by-BAC clone and whole genome shotgun) approaches.