US5565350A
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Compounds and methods for site directed mutations in eukaryotic cells
Assignee: Thomas Jefferson University
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Filing: 1994-12-09
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Granted: 1996-10-15
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Expires: 2014-12-09
Abstract
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.
Inventors
- 👤 Eric B. Kmiec
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 00:58
CPC Classifications
C; C07; C07H; C07H21/00; C07H21/02; A; A01; A01K; A01K67/00; A01K67/027; A01K67/0275; C07H21/04; C07K; C07K14/00; C07K14/82; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/87; C12N15/90; C12N15/902; C12N15/907; A01K2217/00; A01K2217/05
Claims Analysis
Total Claims: 31
Claim 1
Independent Claim
119 words
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.
Claim 2
Dependent Claim
119 words
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.
Claim 3
Dependent Claim
21 words
The nucleic acid of claim 1 in which the second strand contains no nucleotides comprised of a 2'-O or 2'-OMe ribose.
Claim 4
Dependent Claim
27 words
The nucleic acid of claim 2 in which the hybrid-duplex comprises at least 9 contiguous nucleotides comprised of a 2'-O ribose flanked by nucleotides comprised of 2'-deoxyribose.
Claim 5
Dependent Claim
59 words
The nucleic acid of claim 2 in which the first strand comprises two regions of contiguous nucleotides comprised of a 2'-O or 2'-OMe ribose that form two regions of hybrid-duplex, each hybrid-duplex region having at least 4 base pairs of length, and an interposed region of at least 1 base pair of homo-duplex disposed between the hybrid duplex regions.
Claim 6
Dependent Claim
58 words
The nucleic acid of claim 2 in which the first strand comprises two regions of nucleotides comprised of a 2'-O or 2'-OMe ribose that form two regions of hybrid-duplex, each hybrid-duplex region having at least 8 base pairs of length, and an interposed region of at least 4 base pairs of homo-duplex disposed between the hybrid duplex regions.
Claim 7
Dependent Claim
22 words
The nucleic acid of claim 5 in which the interposed region of homo-duplex consists of between 4 and 50 2'-deoxyribose base pairs.
Claim 8
Dependent Claim
22 words
The nucleic acid of claim 5 in which the interposed region of homo-duplex consists of between 30 and 1,000 2'-deoxyribose base pairs.
Claim 9
Dependent Claim
32 words
The nucleic acid of claim 2 in which the 5' and 3' ends are Watson Crick paired to adjacent bases of a complementary strand in the region of Watson-Crick paired nucleic acid.
Claim 10
Dependent Claim
31 words
The nucleic acid of claim 5 in which the 5' and 3' ends are Watson-Crick paired to adjacent bases of a complementary strand in the region of Watson-Crick paired nucleic acid.
Claim 11
Dependent Claim
31 words
The nucleic acid of claim 6 in which the 5' and 3' ends are Watson-Crick paired to adjacent bases of a complementary strand in the region of Watson-Crick paired nucleic acid.
Claim 12
Dependent Claim
31 words
The nucleic acid of claim 7 in which the 5' and 3' ends are Watson-Crick paired to adjacent bases of a complementary strand in the region of Watson-Crick paired nucleic acid.
Claim 13
Independent Claim
76 words
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.
Claim 14
Independent Claim
76 words
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.
Claim 15
Dependent Claim
28 words
The method of claim 12 wherein the nucleus is a pronucleus of an ovum and the mixed ribo-deoxyribonucleic acid vector is introduced into the pronucleus by direct injection.
Claim 16
Dependent Claim
23 words
The method of claim 12 wherein the nucleus is a nucleus of an embryonic stem cell and the introduction is by direct injection.
Claim 17
Independent Claim
75 words
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.
Claim 18
Independent Claim
75 words
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.
Claim 19
Dependent Claim
12 words
The method of claim 15 wherein the cell is a mammalian cell.
Claim 20
Dependent Claim
13 words
The method of claim 15 wherein the cell is a yeast or fungus.
Claim 21
Dependent Claim
31 words
The nucleic acid of claim 1, in which the 5' and 3' ends are Watson-Crick paired to adjacent bases of a complementary strand in the region of Watson-Crick paired nucleic acid.
Claim 22
Dependent Claim
16 words
The nucleic acid of claim 18, which comprises between 15 and 50 base pairs of hybrid-duplex.
Claim 23
Dependent Claim
17 words
The nucleic acid of claim 19, in which the second strand comprises the 5' and 3' ends.
Claim 24
Dependent Claim
15 words
The nucleic acid of claim 8, which comprises between 15 and 50 base pairs hybrid-duplex.
Claim 25
Dependent Claim
17 words
The nucleic acid of claim 21, in which the second strand comprises the 5' and 3' ends.
Claim 26
Dependent Claim
16 words
The nucleic acid of claim 9, which comprises between 15 and 50 base pairs of hybrid-duplex.
Claim 27
Dependent Claim
17 words
The nucleic acid of claim 23, in which the second strand comprises the 5' and 3' ends.
Claim 28
Dependent Claim
16 words
The nucleic acid of claim 10, which comprises between 15 and 50 base pairs of hybrid-duplex.
Claim 29
Dependent Claim
17 words
The nucleic acid of claim 25, in which the second strand comprises the 5' and 3' ends.
Claim 30
Dependent Claim
16 words
The nucleic acid of claim 1, which comprises between 15 and 50 base pairs of hybrid-duplex.
Claim 31
Dependent Claim
16 words
The nucleic acid of claim 2, which comprises between 15 and 50 base pairs of hybrid-duplex.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-10-15 | 2000-04-15 | 2000-10-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-10-15 | 2004-04-15 | 2004-10-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-10-15 | 2008-04-15 | 2008-10-15 | $7700.00 | Window Closed / Lapsed |