The invention is based on the reaction of recombinagenic oligonucleotides in a
cell-
free system containing a cytoplasmic
cell extract and a test duplex
DNA on a
plasmid. The reaction specifically converts a
mutant kanr
gene to recover the
resistant phenotype in transformed MutS, RecA deficient
bacteria and allows for the rapid and quantitative comparison of recombinagenic oligonucleobases. Using this
system a type of Duplex Mutational Vector termed a
Heteroduplex Mutational Vector, was shown to be more active in than the types of mutational vectors heretofore tested. Further improvements in activity were obtained by replacement of a tetrathymidine
linker by a
nuclease resistant
oligonucleotide, such as
tetra-2'-O-methyl-
uridine, to link the two strands of the Duplex Mutational Vector and removal of the
DNA-containing intervening segment. The claims concern Duplex Mutational Vectors that contain the above improvements. In an alternative embodiment the claims concern a reaction mixture containing a recombinagenic oligonucleobase, a
cell-free
enzyme mixture and a duplex
DNA containing a target sequence. In yet an alternative embodiment, the invention concerns the use of such mixture to test improvements in recombinagenic oligonucleobases, as well as to test the effects of compounds on the activity of the cell-free
enzyme mixture and also to make specific changes in the target DNA sequence.