Rapid Construction of Sequencing Templates by Random Insertion of Antibiotic Resistance Genes | Zendy

Е. Н. Набирочкина | Zendy; Sofia G. Georgieva | Zendy; А. Н. Краснов | Zendy; Aleksey V. Soldatov | Zendy

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Rapid Construction of Sequencing Templates by Random Insertion of Antibiotic Resistance Genes

Author(s) -

Е. Н. Набирочкина,

Sofia G. Georgieva,

А. Н. Краснов,

Aleksey V. Soldatov

Publication year - 2002

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/02322st04

Subject(s) - plasmid , biology , genetics , dna , gene , insertional mutagenesis , mutagenesis , template , mutant , restriction enzyme , population , dna sequencing , computational biology , computer science , medicine , environmental health , programming language

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.

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