Abstract: 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.

Abstract: The invention is based on the reaction of Duplex Mutational Vector in a cell-free system containing a cytoplasmic cell extract and a test plasmid. The reaction specifically converts a mutant kan.sup.r gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria. Using this system a type of Duplex Mutational Vector termed a Non-Chimeric Mutational Vector, having no RNA:DNA hybrid-duplex is shown to be an effective substrate for eukaryotic enzymes. The invention concerns the use of Non-Chimeric Mutational Vectors protected from 3' exonuclease attack in eukaryotic cells. Such protection can be conferred 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 recombinagenic oligonucleobase.

Abstract: The invention concerns methods of introducing specific alterations in the genome of cells that have been removed from a subject suffering from a medical condition that is the result of a genetic lesion. The specific alteration is designed to correct the genetic lesion. The method comprises introducing an oligonucleotide compound, containing ribonucleotides and deoxyribonucleotides, into the cells and thereafter reintroducing the cells into the subject. Specific types of cells include hematopoietic stem cells and hepatocytes. The ribonucleotides of the compound can have 2-substituents that enhance their resistance to RNase.