Random Transposon Mutagenesis of Large DNA Molecules in Escherichia coli

Mutagenesis of large plasmids, such as bacterial artificial chromosomes (BACs) or P1-based artificial chromosomes (PACs), can be difficult for various reasons. Because of their large size (up to 300 kbp), unique restriction enzyme sites are usually not available. Even if they are, modifications based on restriction digest and ligation are mastered only by scientists with extensive experience in handling large DNA molecules. Moreover, sequence information is not available for many BACs and PACs derived from eukaryotic genome libraries, which further complicates mutagenesis.