Abstracts: 

 Physical Mapping 



Human Genome Center, Lawrence Berkeley Laboratory 



Charles R. Cantor, C. Bustamante. M. Esposito, J. Gingrich, S. Levene, M. Maestre, 

 R. Mortimer. M. Saimeron, C. L. Smith, and M. Stoneking 

 Human Genome Center, Lawrence Berkeley Laboratory, Berkeley, CA 94720 

 (415)486-6800, (FTS) 451 -6Sn() 



Researchers at the Human Genome Center at Lawrence Berkeley Laboratory (LBL) are 

 developing methodologies needed to complete a physical map and an ordered library of 

 the human genome. A top-down approach will be used by developing yeast artificial 

 chromosome libraries prepared both from total genomic DNA and from specific 

 physically isolated human chromosomes. The immediate goal is to integrate the 

 physical map, as it is developed, with the genetic map by defining the sites on the 

 physical map of cloned and genetically localized genes of specific significance to the 

 Office of Health and Environmental Research (OHER) mission. Several methods for 

 constructing the ordered map will be investigated, some of which include using junction 

 fragments, determining fragment overlap by restriction maps, and employing 

 recombination among artificial chromosomes. 



Brief abstracts of the individual projects are listed below. 



Optimization of Yeast Artificial Chromosome (YAC) Mapping (M. Esposito, J. 

 Gingrich, R. Mortimer, and C. L. Smith) — The use of larger DNA fragments means 

 that fewer fragments need to be ordered into a map: consequently, the initial major 

 focus has been to obtain clones containing large fragments of DNA from chromosome 

 21. The most promising avenue appears to be the use of YAC vectors. Different 

 strategies for producing YAC clones are currently being evaluated and optimized. Since 

 the starting material being used for these clones is a hybrid cell line consisting of human 

 chromosome 21 in a background of mouse chromosomes, a major part of the effort is 

 devoted to methods for identifying YAC clones that contain human DNA. These clones 

 are expected to comprise only 1-2% of the total YAC clones from this cell line. The 

 polymerase chain reaction is being evaluated as a new approach to this problem. This 

 identification strategy is based on the expectation that only those clones containing 

 human DNA would be amplified from a known human DNA sequence primer. A 

 method to link up YACs with overlapping sequence infonnation by using 

 recombination is under development, as are new methods for improved DNA 

 transfection into yeast. 



New Mapping Methods 



Sequence Matching (C. L. Smith and M. Stoneking) — To construct a map, a means of 

 uniquely identifying each DNA fragment is necessary; the strategy at LBL is to use 

 DNA sequences from the ends of the fragments. Knowing a small sequence (50-100 

 bp) from each end of a large DNA fragment will permit each unique fragment to be 

 identified. Furthermore, matching these DNA sequences with similarly sized DNA 



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