Patent Evaluation: Library Screening Method: A Novel Approach for Cloning DNA

Abstract

SummaryNovelty: A cloning strategy and vectors used to carry out the cloning are described. These may facilitate the isolation of clones containing sequences homologous to another sequence, by using homologous recombination in yeast.Biology: A library of DNA fragments is cloned into a YAC (yeast artificial chromosome) vector in a yeast strain. A DNA fragment or gene cloned into a bacterial vector, which will not replicate in yeast, is introduced in a linear state into the YAC library and any clone which contains a sequence homologous to the linearized DNA can undergo homologous recombination with the YAC clone. The system is set up so that recombinants containing DNA sequences to one side of the YAC clone can be selected on 5-fluoro-orotic acid plates lacking arginine. Clones with sequences on the other side of the YAC are selected on media containing α-amino adipate lacking tryptophan. Since YAC vectors can contain very large regions of DNA, whole genes may be isolated in a YAC vector by only having a small portion of the DNA or a cDNA clone. This may make chromosome walking a more simple process for very large complex human genes.