Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.
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.
Saltman
D L
DL
Department of Molecular Genetics, Genelabs Incorporated, Redwood City, CA 94063.
Dolganov
G M
GM
Pearce
B S
BS
Kuo
S S
SS
Callahan
P J
PJ
Cleary
M L
ML
Lovett
M
M
D001483:Base Sequence; D015139:Blotting, Southern; D002895:Chromosomes, Human, Pair 5; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D004247:DNA