ASSEMBLY OF ORDERED CONTIGS OF COSMIDS SELECTED WITH YACS OF HUMAN-CHROMOSOME-13

We have developed an efficient method for assembling ordered cosmid co ntigs aligned to mega-YACs and midi-YACs (average insert sizes of 1.0 and 0.35 Mb, respectively) and used this general method to initiate hi gh-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13 enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtain ed from corresponding CEPH total. human YAC libraries by selecting col onies with inter-Alu PCR probes derived from Chr 13 monochromosomal ce ll hybrid DNA These sublibraries were arrayed on filters at high densi ty. In our approach, the MYAC 13 sublibrary is screened by hybridizati on with cytogenetically assigned Chr 13 DNA probes to select one or a small subset of MYACs. Inter-Alu PCR products from each MYAC are then hybridized to the MYAC and mYAC sublibraries to identify overlapping Y ACs and to an arrayed Chr 13-specific cosmid library to select corresp onding cosmids. The set of selected cosmids, gridded on filters at hig h density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprob es from each cosmid of the arrayed set (''cosmid matrix cross-hybridiz ation''). From these data, cosmid contigs are assembled by a specifica lly designed computer program. Application of this method generates co smid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sit es to position densely spaced sequence-tagged sites. (C) 1994 Academic Press, Inc.