Rapid construction of sequencing templates by random insertion of antibiotic resistance genes.
We describe a novel and handy method for generating a population of templates for sequencing. The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I. The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid. DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background. We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants.
Nabirochkina
Elena N
EN
Institute of Gene Biology, RAS Moscow, Russia.
Georgieva
Sofia G
SG
Krasnov
Aleksey N
AN
Soldatov
Aleksey V
AV
D007613:Kanamycin Resistance / Q000235:genetics*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic