[The method of template construction for sequencing extended DNA fragments].
A rapid and convenient method was proposed for constructing insertional mutants in the sequencing of extended DNA fragments. The gist is insertion of an antibiotic resistance gene in plasmid DNA digested with DNase I. DNase I provides for a uniform distribution of insertion sites along the plasmid, and the background is low owing to antibiotic-based selection. The method requires neither high quality nor large amounts of plasmid DNA (which is especially important with low-copied plasmids), yields the results independent of the plasmid nucleotide sequence, and allows highly accurate analysis and ordering of the insertional mutants.
Ermolaeva
M A
MA
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 117334 Russia.
Soldatov
A V
AV
Nabirochkina
E N
EN
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000378:metabolism; D005821:Genetic Techniques; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D000900:Anti-Bacterial Agents; D003850:Deoxyribonuclease I