Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones

Abstract

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.