A novel procedure for selective cloning ofNotI linking fragments from mammalian genomes

Abstract

A novel procedure has been developed for selective cloning of NotI linking fragments from mammalian genomes. Since the majority of the NotI sites in mammalian genomes are considered to be localized in so-called HTF (Hpall tiny fragment) islands, an HTF library was constructed as an initial step to enrich the Notl sites. The plasmid DNAs were isolated en masse from the HTF library and digested with Notl. Linearized plasmid DNAs derived from Notl linking clones were efficiently separated from undigested circular DNAs by an unique pulsed field polyacrylamide gel electrophoresis (PF-PAGE). The linear DNAs were eluted from the gel, recircularizcd with T4 DNA ligase and introduced into E.coli cells. About 95% of the transformants were found to contain NotI linking fragments. The procedure will thus provide a simple and useful way of collecting Notl linking fragments for long range physical mapping of mammalian genomes.