Cross-screening: a new method to assemble clones rapidly and unambiguously into contigs.
We have developed a new procedure that relies on an array of cross-hybridization tests to order a set of random clones into a contig. The method, called cross-screening, uses each clone as a target and its end sequences as probes, in a matrix of reciprocal cross-hybridization tests performed on a single blot. The relationships among the clones are determined rapidly from the pairwise tests, allowing clone order to be determined directly. We have applied this technique to DNAs from a set of overlapping lambda clones from Drosophila chromosome 4. The location and orientation of each clone derived from the cross-screening data was that expected from the map assembled from overlapping restriction sites and chromosomal walking. The procedure provided additional information on a previously unknown, internally repeated DNA sequence. To demonstrate the general utility of the procedure, we have applied it to a previously described clone set within a contig in region 22q12 of human chromosome 22. The correct relative position and orientation of these clones were derived from the cross-screening data without knowledge of, or reference to, any nucleotide sequence or restriction site analysis of the DNA concerned. The cross-screening procedure is fast, economical, and robust and allows clone overlaps to be determined efficiently, with minimal interference from repeated DNA sequences. This new procedure is specifically designed for small groups of overlapping clones (tens to hundreds) and should facilitate the ordering of subclone libraries derived from small chromosomes or the large cloned inserts carried in YAC, BAC, and P1 vectors.
Locke
J
J
Department of Biological Sciences, University of Alberta, Edmonton, Canada. john_locke@biology.ualberta.ca
Rairdan
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McDermid
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Nash
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Pilgrim
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Bell
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Roy
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Hodgetts
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D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002892:Chromosomes, Human, Pair 22; D003001:Cloning, Molecular; D003360:Cosmids; D004331:Drosophila melanogaster / Q000235:genetics; D005807:Genes, Overlapping; D006801:Humans; D012091:Repetitive Sequences, Nucleic Acid