Blocking the ends of transforming DNA enhances gene targeting in Dictyostelium.

Eukaryotic gene targeting by means of gene replacement vectors is often complicated by unwanted plasmid insertion events involving the ends of transforming DNA molecules. These undesirable and often multiple insertions occur both randomly (i.e. non-homologously) and at the targeted locus. By blocking the 3' ends of transforming DNA with 2'3' dideoxynucleotides, we have reduced the frequency of end-mediated DNA insertion in Dictyostelium amoebae. As a result, only one copy of the selectable gene is introduced at the target locus to achieve a precise gene disruption.