High efficiency vectors for cosmid microcloning and genomic analysis.
We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.
Molecular Genetics Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.
G A
GA
Evans
K
K
Lewis
B E
BE
Rothenberg
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015347:RNA Probes; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping
D015347:RNA Probes; D004247:DNA