Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.