Top-down construction of an ordered Schizosaccharomyces pombe cosmid library.
A very rapid and efficient method for sorting and ordering large numbers of clones is presented. This top-down mapping approach divides the entire ordering problem into many smaller tasks and analyzes in parallel a gridded membrane array of clones by hybridization with probe pools. The strategy was tested on a 15-fold-coverage Schizosaccharomyces pombe cosmid library. About 1600 clones were assigned to chromosomes and to regions defined by the Not I and Sfi I restriction maps. Then, the clones were ordered into 20 contigs, which is consistent with statistical expectations for the degree of genome coverage used. The parallel ordering of clones and the computer-based analysis of digitized images make this approach very efficient; it is about 8-fold faster than existing methods. Only 61 hybridizations were needed to order 1600 clones.
Grothues
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Cantor
C R
CR
Smith
C L
CL
D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal