Methods of targeting a chromosomal gene sequence in a eukaryotic cell

The present invention relates to targeting of genes, and more particularly to targeting of chromosomal gene sequences in eukaryotic cells. The invention provides in one embodiment a composition which is a linear DNA molecule having a desired replacement sequence, and second and third sequences substantially homologous to non-identical portions of the gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence and the distal ends having a terminating nucleotide analog at each end of the molecule. Another embodiment of the invention provides methods, by blocking the 3′ ends of transforming DNA with 2′3′ dideoxynucleotides, to reduce the frequency of end-mediated DNA insertion. These methods introduce only one copy of the selectable gene at the target locus to achieve a precise gene disruption, reducing or eliminating undesirable and multiple insertions that occur both non-homologously and at the targeted locus.