Compounds and methods for site directed mutations in eukaryotic cells
AssigneeThomas Jefferson University
InventorEric B. Kmiec
Life of this patent
Estimating the term…
Abstract
The present invention concerns a polynucleotide having both ribonucleotides and deoxyribonucleotides in a first strand and solely deoxyribonucleotides in a second strand; wherein the strands are Watson-Crick paired and are linked by an oligonucleotide so that the polynucleotide has at most a single 3' and a single 5' end. These ends can be ligated so that the polynucleotide is a single continuous circular polymer. The polynucleotide can be used to induce specific alterations in targeted genes.
Claims28
A mixed ribo-deoxyribonucleic acid having at most one 3' end and one 5' end, which nucleic acid further comprises:
- a) at least one region of contiguous unpaired bases disposed so that the unpaired region deparates the nucleic acid into a first strand and a second strand;
- b) connected by said region of contiguous unpaired bases a region of Watson-Crick paired nucleic acid of at least 15 base pairs in length, in which bases of the first strand correspond to bases of the second strand, and in which:
- c) the first strand comprises a region of at least three contiguous nucleotides comprised of a 2'-O or 2'-OMe ribose, which form a hybrid-duplex within the region of Watson-Crick paired nucleic acid.
A method of introducing a predetermined alteration in a target sequence of the genome of a cultured cell, which cell contains a nucleus, which comprises the steps of:
- a) providing mixed ribo-deoxyribonucleic acid vector having two regions homologous with a target sequence and a heterologous region, disposed there between, encoding the alternation;
- and
- b) maintaining the mixed ribo-deoxyribonucleic acid vector within the nucleus of the cultured cell,
- whereby the alteration is introduced into the target sequence.
A method of obtaining a cultured cell population having altered characteristics which comprises the steps of:
- a) providing a mixed ribo-deoxyribonucleic acid vector having two regions homologous with a target sequence and a heterologous region, disposed there between, encoding the alteration;
- b) maintaining the mixed ribo-deoxyribonucleic acid vector within the nucleus of the cultured cell whereby the chimeric vector and the target sequence recombine and the cell is transformed;
- and
- c) selecting the transformed cells.
Dependencies are inferred from the claim text.