Construction and use of human chromosome jumping libraries from NotI-digested DNA

Abstract

A basic difficulty in the molecular analysis of genes identified by mutations in the mammalian genome is the need to cover genetic distances corresponding to several hundred kilobases or more by molecular techniques like chromosome walking. In chromosome jumping, this limitation is overcome by the deletion of all but the extreme ends of large DNA molecules before cloning. We describe here the construction and characterization of a NotI 'jumping library' from human DNA. To characterize this library, random clones were analysed by restriction mapping. Clones carrying unique end fragments were characterized further by hybridization to Southern blots of NotI-cleaved human DNA separated on pulsed field gradient (PFG) gels. As a first step in a directional walk, the library was screened with a clone containing a NotI site cleaved in genomic DNA ('NotI linking clone') localized to the distal third of the short arm of human chromosome 4 (A.-M.F. & T.P., unpublished data). Starting and end points of two identified clones were positioned within a restriction map covering 850 kilobases.