Assembly of ordered contigs of cosmids selected with YACs of human chromosome 13.
We have developed an efficient method for assembling ordered cosmid contigs 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 high-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13-enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtained from corresponding CEPH total human YAC libraries by selecting colonies with inter-Alu PCR probes derived from Chr 13 monochromosomal cell hybrid DNA. These sublibraries were arrayed on filters at high density. In our approach, the MYAC 13 sublibrary is screened by hybridization 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 YACs and to an arrayed Chr 13-specific cosmid library to select corresponding cosmids. The set of selected cosmids, gridded on filters at high density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprobes from each cosmid of the arrayed set ("cosmid matrix cross-hybridization"). From these data, cosmid contigs are assembled by a specifically designed computer program. Application of this method generates cosmid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sites to position densely spaced sequence-tagged sites.
Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
S G
SG
Fischer
E
E
Cayanis
J J
JJ
Russo
I
I
Sunjevaric
B
B
Boukhgalter
P
P
Zhang
M T
MT
Yu
R
R
Rothstein
D
D
Warburton
I S
IS
Edelman
D001483:Base Sequence; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D002882:Chromosomes, Human, Pair 13; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016324:Sequence Tagged Sites
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA