Gene Libraries for Each Human Chromosome: 

 Construction and Distribution 



Marvin A. Van Dilla, Pieter de Jong, Barbara Trask. Anthony V. Carrano, Joe Gray, 



Kathy Yokobata, and Ger J. van den Engh 



Biomedical Sciences Division. Lawrence Livennore National Laboratory, Livermore, 



CA 94550 



(415) 422-5662, FTS 532-5662 



Tiie goal of the National Laboratory Gene Library Project (NLGLP) is the production of 

 chromosome-specific human gene libraries and their distribution to the scientific 

 community for the diagnosis and study of genetic disease, determination of the structure 

 and function of genes, and for the physical mapping of chromosomes. This cooperative 

 project employs the flow-sorting and molecular-cloning expertise at the Los Alamos 

 and Lawrence Livermore national laboratories. The specific aim of Phase I of the 

 project was the production of complete digest libraries from each of the human 

 chromosomal types purified by flow sorting. The bacteriophage lambda vector used was 

 Charon 21 A, which has both EcoR 1 and Hind 111 insertion sites accommodating human 

 DN A fragments 0-9. 1 kb m size. Each laboratory has produced a complete set of 

 chromosome-specific libraries, LANL with EcoR I and LLNL with Hind 111. Library 

 purity ranges from nearly 100% for good chromosome preparations and favorably 

 placed peaks in the flow karyotype to about 50% for some early preparations from cell 

 lines with unfavorably placed peaks. The libraries are deposited in a repository at the 

 American Type Culture Collection ( ATCC). Rockville, Maryland; about 2400 aliquots 

 have been distributed to over 500 laboratories worldwide. All Livennore libraries have 

 been subcloned into the plasmid vector Bluescribe (Stratagene, La Jolla, California), 

 facilitating both the use of the DNA probes and the preparation of RNA end probes. 



Phase 11, the construction of libraries with large inserts in lambda replacement vectors 

 (accept about 9-23 kb) and in cosmid vectors (accept about 33-46 kb), is under way. 

 These large insert libraries have characteristics that are better suited to basic studies of 

 gene structure and function, organization of genes on chromosomes, and ordering of 

 cloned sequences. The Phase II strategy is to split the genome cloning responsibility 

 between the two laboratories [i.e., Livermore will clone 12 chromosomal types (1. 2, 3, 

 7,9, 11, 12, 18, 19, 21.22, and Y), and Los Alamos will clone the other 12(4,5,6,8, 

 10, 13, 14, 15, 16. 17, 20, and X)]. In this way, each chromosomal type will be cloned 

 into both lambda and cosmid vectors. Livermore is using the lambda vector Charon 40 

 (accepts 10-25 kb inserts) and, more recently, lambda GEMl 1, which has about the 

 same acceptance range as Charon 40 but is particularly suited for the manipulations 

 required to efficiently clone, map, sequence, and "walk"" along contiguous segments of 

 genomic DNA. At Livennore, the cosmid vector is Lawrist 5 (accepts inserts of 

 34-46 kb). which has the same advantageous features for users as lambda GEMl 1 and 

 is double the insert size. The cloning procedures (more complicated than for Phase I) 

 have now been worked out, and we have constructed two large Charon 40 libraries for 



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