Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

• Published in: Gene Vol. 71; no. 2; pp. 391 - 400
• Main Authors: Bowden, Donald W., Müller-Kahle, Hans, Fulton, Thomas R., Gravius, Thomas C., Barker, David F., Donis-Keller, Helen
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 30.11.1988; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biotechnology; Blotting, Southern; Chromosome Mapping; Cloning, Molecular; cosmid vector; Cosmids; DNA - genetics; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic Linkage; Genetic technics; Genetic Vectors; human genomic DNA; Humans; Methods. Procedures. Technologies; mini-libraries; Molecular cloning; Nucleic Acid Hybridization; Nucleic Acids - analysis; Pedigree; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Recombinant DNA; restriction fragment length polymorphisms; Restriction Mapping; Retinol-Binding Proteins - genetics; Southern-blot hybridization; c; ch; restriction fragment length polymorphisms; dNTP; mini-libraries; Southern-blot hybridization; cM; IL-2; Recombinant DNA; human genomic DNA; bp; Ap; cfu; IRBP; kb; cDNA; cosmid vector; RFLP; Human; Molecular hybridization; Linkage; Family study; Cosmid; Chromosome walking; Gene library; Method; Screening; Restriction fragment length polymorphism; Complementary DNA; C10-Chromosome; Genetic engineering; Molecular cloning; Southern blotting
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(88)90056-X

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.