Eukaryotic use of improved chimeric mutational vectors
US6573046B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Oct 28, 1999 |
| Grant date | Jun 3, 2003 |
| Priority date | — |
| Expiry date | Oct 28, 2019 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/102
- WIPO fieldBiotechnology
- WIPO sectorChemistry
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. 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 mix…
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.