Abstracts: 

 Physical Mapping 



Developing a Physical Map of Human Chromosome 22 

 Using PACE Electrophoresis and Large Fragment Cloning 



Melvin I. Simon. Bruce Birren. and Hiroaki Shizuya 



Biology DiviMon, California Institute ot Technology. Pasadena. CA 91 106-4107 



(818)356-3944 



The goal of this project is to derive a set of overlapping clones covering human 

 chromosome 22. Much of the work involves the development of new or improved 

 methods for cloning large DNA fragments, and for handling, analyzing, and 

 overlapping these clones. To create an overlapping clone map of human chromosome 

 22. a set of new bacterial artificial chromosome (BAC) vectors will be developed that 

 should support the cloning of large DNA fragments about 200 kb in length. These BAC 

 vectors are based on the E. coli F-factor and will be constructed to contain promoters 

 for walking, a multiple cloning site, a cos site for cleavage reactions, rare-cutting sites 

 surrounding the insert, and two selectable markers. A library of chromosome 22 will be 

 constructed in these vectors, in modified YAC vectors, and in cosmids. Source DNA for 

 the libraries will come from hamster/human hybrid cells that contain either intact or 

 deleted chromosome 22. For the hybrids with deletions, the pulse alternating current 

 electrophoresis (PACE) system will be used to separate the deleted chromosome. 

 Human clones will be selected from the libraries by screening with total human DNA. 

 Fingerprints of the clones from the different vector systems will be achieved by partially 

 digesting the cloned DNA and labeling the cos sites with either radioactive or 

 fluorescent tags. The cos sites will be cut with terminase and labeled by a hybridization- 

 ligation reaction. Since each cos end has a different sequence, different oligos can be 

 ligated to each site and a partial-digest map can be created from each end of the clone. 

 The use of fluorescent tags attached by ligation allows the simultaneous use of different 

 fluorochromes at each cos site while separate restriction analysis would be done for 

 radiolabeled oligonucleotides. Detection of the restriction fragments would be 

 performed on the PACE pulsed-field gel electrophoresis system. Based upon the partial 

 digest data, computer algorithms will construct the overlap map. 



74 



