Second-generation approach to the construction of yeast artificial-chromosome libraries

Abstract

We describe an improved method for construction of yeast artificial-chromosome (YAC) libraries that contain large inserts of foreign DNA. The procedure consists of seven steps: (i) preparation of human DNA in agarose beads; (ii) partial digestion of the DNA with EcoRI; (iii) electrophoretic elimination of the smaller partial-digest fragments; (iv) ligation of the EcoRI fragments with vector arms in molten agarose; (v) hydrolysis of agarose with agarase; (vi) fractionation of the recombinant molecules by sucrose-gradient centrifugation; and (vii) transformation of yeast. More than 7000 colonies were obtained starting with 15 micrograms of human DNA, which was fractionated on a single sucrose gradient. The average size of these YACs was approximately 380 kb. It is estimated that the total length of human DNA present in the clones corresponds to 80% of the length of the human haploid genome. The results of screening the clones for a number of single-copy genes indicate that the clones reflect a nearly random sampling of the human genome. The efficiency of the cloning is sufficient to support the construction of multihit libraries for the human genome or for the genomes of other higher organisms.