Construction of a Human MluI YAC Library

Abstract

We describe a cloning strategy for the construction of a human genomic library in yeast artificial chromosomes (YACs) based on complete digestion of high-molecular-weight DNA with the infrequently cutting restriction enzyme Mlu I. Cloning of MluI fragments in the vector pYAC-RC and one subsequent size fractionation by preparative pulsed-field gel electrophoresis yielded a library with average insert sizes of 600 kb. Ninety-seven percent of the colonies were recombinant. An additional size fractionation of MluI fragments prior to ligation had no significant influence on the size of the YACs. The library currently consists of 5000 clones, which is the equivalent of one human genome. Nineteen percent of the YACs were larger than 1.2 Mb. Since smaller MluI fragments are lost during sizing, we also performed cloning without size fractionation. Only 20% of the colonies were recombinant, probably due to unligated vector fragments that were present during the transformation.