CONSTRUCTION OF A HUMAN DNA LIBRARY IN A CIRCULAR CENTROMERE-BASED YEAST PLASMID

The construction of a human DNA library in a centromere-based circular yeast plasmid is described. The vector contains the yeast CEN3 sequen ce, the URA3 gene for propagation in yeast and a hygromycin-resistance gene (Hy(R)) for selection in mammalian cells, The library consists o f 64 000 members with an average insert size of 150 kb, with some memb ers containing inserts of >1 Mb. We calculate that the library contain s three human genome equivalents of DNA, Clones can be identified by a PCR-based screening of DNA pools from individual colonies that have b een stored in microtiter wells.