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.
Bingle
W H
WH
Department of Microbiology, Univ. of British Columbia, Vancouver, Canada.
Smit
J
J
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005819:Genetic Markers; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids
D005819:Genetic Markers; D004247:DNA; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI