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-hyb ridization tests to order a set of random clones into a contig. The me thod, called cross-screening, uses each clone as a target and its end sequences as probes, in a matrix of reciprocal cross-hybridization tes ts 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 se t of overlapping lambda clones from Drosophila chromosome 4. The locat ion and orientation of each clone derived from the cross-screening dat a was that expected from the map assembled from overlapping restrictio n sites and chromosomal walking. The procedure provided additional inf ormation 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 knowl edge of, or reference to, ally 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 eff iciently, 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 subcl one libraries derived from small chromosomes or the large cloned inser ts carried in YAC, BAC, and P1 vectors.