A Method of Tagging Specific-purpose Linkers with an Antibiotic-resistance Gene for Linker Mutagenesis Using a Selectable Marker

A method is described for tagging double-stranded linkers with an antibiotic-resistance gene permitting the direct selection of specific linker insertions during random linker-insertion mutagenesis; after selection, the antibiotic-resistance marker is removed leaving the linker-encoded restriction site in the target gene. The method is simple, relying on commercially available linkers and DNA-modifying enzymes, but retains considerable target site flexibility. The tagged linkers can be inserted at blunt-ended sites created in the target gene with DNase I or at sites created by restriction enzyme digestion leaving blunt ends, 5'-CG or 5'-GATC extensions. The advantages of such an approach over the use of standard antibiotic-resistance gene cartridges is discussed.