Rapid method for construction of yeast artificial chromosome human DNA libraries involving the trapping of cells in agarose films

摘要

>A simple method for molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cell in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector in situ YAC construction [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment. YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.