Abstracts: 

 Physical Mapping 



Physical Maps of Human Chromosomes: Methods 

 Development and Applications 



Anthony V. Carrano. Elbert W. Branscomb, Pieter J. de Jong, Emilio Garcia, 



Harvey W. Mohrenweiser, and Thomas Slezak 



Biomedical Sciences Division, Lawrence Livemiore National Laboratory, 



Livemiore, CA 94550 



(415) 422-5698. FTS 532-5698 



The initial goal of this project is to create physical maps of human chromosomes and to 

 correlate them with the genetic map. The physical maps will consist of overlapping 

 cloned DNA fragments (contigs) contained in phage, cosmid, and yeast vectors, all of 

 which span the chromosomes. The project is multidisciplinary, and its components are 

 synergistic. In the past two years, progress has been made in several areas. We 

 constructed new or modified existing vectors to ( 1 ) facilitate cloning small amounts of 

 DNA in cosmids, (2) clone Not I linking probes in lambda and plasmids, and (3) clone 

 large fragments of DNA as yeast artificial chromosomes ( YACs). Several of the cosmid 

 vectors have been transferred to industry. The cosmid vectors were used to construct 

 chromosome- 1 9-specific libraries from tlow-sorted chromosomes and from a 

 monochromosomal hybrid. About 10.000 cosmids (about sixfold redundancy) have 

 been arrayed in microtiler trays to form a reference library for chromosome 19. We used 

 the new plasmid and lambda vectors to create a No! 1 linking library of chromosome 19 

 and have initially isolated about 30 clones. We are currently expanding and character- 

 izing libraries of chromosome 19 in YAC and half-YAC vectors. To construct a set of 

 cosmid contigs for chromosome 19, we developed an automated fluorescence-based 

 strategy for fingerprinting each clone. For this procedure, a robotic system is used to 

 attach fluorophores to the ends of restriction fragments from each cosmid clone. 

 Fragment lengths are determined by using a commercially available laser scanning 

 device to acquire electrophoretic mobility data in real time. Up to four different 

 fluorophores (i.e., four clones) can be run in each gel lane. In the present configuration, 

 this permits us to analyze up to 48 cosmids per gel run. We developed software to 

 process the acquired fluorophore signals, convert the signal data to restriction fragment 

 lengths for each cosmid, and use the fragment length data to compute a statistical 

 measurement of overlap between cosmids. Several thousand cosmids have been 

 processed to date. We have established 6 cosmid contigs that span approximately 600 

 kb of chromosome 14 and have over 200 contigs for chromosome 19. Five of the 

 chromosome- 19 contigs represent known gene loci, and the others are located 

 throughout the chromosome. Contigs are validated by restriction fragment digests and/ 

 or by in situ hybridization to metaphase chromosomes. By using large-fragment 

 analysis from pulsed-field gels to close the region of chromosome 19 containing three 

 DNA repair genes and the myotonic dystrophy locus, we discovered that two of the 

 DNA repair genes lie within 260 kb of each other. Finally, we devised a technique, 

 based upon PCR amplification of DNA, to isolate region-specific probes located 

 between human Alii repetitive sequences. These probes are being used to identify those 



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