CONSTRUCTION OF A HUMAN MLUI YAC LIBRARY

We describe a cloning strategy for the construction of a human genomic library in yeast artificial chromosomes (YACs) based on complete dige stion of high-molecular-weight DNA with the infrequently cutting restr iction enzyme MluI. Cloning of MluI fragments in the vector pYAC-RC an d one subsequent size fractionation by preparative pulsed-field gel el ectrophoresis yielded a library with average insert sizes of 600 kb. N inety-seven percent of the colonies were recombinant. An additional si ze fractionation of MluI fragments prior to ligation had no significan t influence on the size of the YACs. The library currently consists of 5000 clones, which is the equivalent of one human genome. Nineteen pe rcent of the YACs were larger than 1.2 Mb. Since smaller MluI fragment s are lost during sizing, we also performed cloning without size fract ionation. Only 20% of the colonies were recombinant, probably due to u nligated vector fragments that were present during the transformation. (C) 1994 Academic Press, Inc.