Patent US6695650B2 successfully refreshed from Google Patents.
US6136601A
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US
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Targeted mutagenesis in living cells using modified oligonucleotides
Assignee: Nanogen Inc
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Filing: 1997-03-26
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Granted: 2000-10-24
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Expires: 2017-03-26
Abstract
Abstract A method for introducing a site-specific mutation into a target polynucleotide sequence is presented. The method involves the use of an oligonucleotide capable of binding to the target sequence, either by triplex formation (mediated by Hoogsteen, reverse Hoogsteen or equivalent base pairing) or by Watson/Crick base pairing (in the presence of a recombinase enzyme). The oligonucleotide of the invention is modified by the covalent attachment of one or more electrophilic groups. When a modified oligonucleotide is bound to its target sequence, the electrophilic group is able to interact with a nearby nucleotide in the target sequence, causing a modification to the nucleotide that results in a change in nucleotide sequence. Compositions used in the practice of the method are also disclosed.
Inventors
- 👤 Rich B. Meyer, Jr.
- 👤 Howard B. Gamper
- 👤 Igor V. Kutyavin
- 👤 Alexander A. Gall
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 15:37
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6839
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
44 words
A modified oligonucleotide for site-directed mutagenesis of a double-stranded nucleic acid molecule comprising one or more mutagens incorporated into a single-stranded oligonucleotide, said oligonucleotide having a sequence that forms a triple-stranded nucleic acid molecule with a target sequence in the double-stranded nucleic acid molecule.
Claim 2
Dependent Claim
44 words
A modified oligonucleotide for site-directed mutagenesis of a double-stranded nucleic acid molecule comprising one or more mutagens incorporated into a single-stranded oligonucleotide, said oligonucleotide having a sequence that forms a triple-stranded nucleic acid molecule with a target sequence in the double-stranded nucleic acid molecule.
Claim 3
Dependent Claim
29 words
The oligonucleotide according to claim 1 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.
Claim 4
Dependent Claim
13 words
The oligonucleotide according to claim 1 wherein the mutagen is an electrophilic group.
Claim 5
Dependent Claim
13 words
The oligonucleotide according to claim 1 wherein the mutagen is an alkylating agent.
Claim 6
Dependent Claim
16 words
The oligonucleotide according to claim 1 wherein two or more mutagens are attached to the oligonucleotide.
Claim 7
Dependent Claim
17 words
The oligonucleotide according to claim 5 wherein two or more alkylating agents are attached to the oligonucleotide.
Claim 8
Independent Claim
64 words
A method for site-directed mutagenesis of a nucleic acid molecule comprising the steps of: a) contacting a modified oligonucleotide to a target sequence of a double-stranded nucleic acid molecule, wherein the modified oligonucleotide comprises one or more mutagens incorporated into a single-stranded nucleic acid that forms a triple-stranded nucleic acid molecule with the target region; and b) thereby mutating the double-stranded nucleic acid molecule.
Claim 9
Independent Claim
64 words
A method for site-directed mutagenesis of a nucleic acid molecule comprising the steps of: a) contacting a modified oligonucleotide to a target sequence of a double-stranded nucleic acid molecule, wherein the modified oligonucleotide comprises one or more mutagens incorporated into a single-stranded nucleic acid that forms a triple-stranded nucleic acid molecule with the target region; and b) thereby mutating the double-stranded nucleic acid molecule.
Claim 10
Dependent Claim
29 words
The method according to claim 7 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.
Claim 11
Dependent Claim
13 words
The method according to claim 7 wherein the mutagen is an electrophilic group.
Claim 12
Dependent Claim
13 words
The method according to claim 7 wherein the mutagen is an alkylating agent.
Claim 13
Dependent Claim
16 words
The method according to claim 7 wherein two or more mutagens are attached to the oligonucleotide.
Claim 14
Dependent Claim
17 words
The method according to claim 7 wherein two or more alkylating agents are attached to the oligonucleotide.
Claim 15
Dependent Claim
18 words
The method according to claim 7 wherein the mutagenesis alters the activity of the double-stranded nucleic acid molecule.
Claim 16
Independent Claim
43 words
A method of producing a mutagenic oligonucleotide comprising the steps of: a) synthesizing an oligonucleotide which forms a triple-stranded nucleic acid molecule with a target sequence of a double-stranded nucleic acid molecule; and b) covalently attaching one or more mutagens to the oligonucleotide.
Claim 17
Independent Claim
43 words
A method of producing a mutagenic oligonucleotide comprising the steps of: a) synthesizing an oligonucleotide which forms a triple-stranded nucleic acid molecule with a target sequence of a double-stranded nucleic acid molecule; and b) covalently attaching one or more mutagens to the oligonucleotide.
Claim 18
Dependent Claim
29 words
The method according to claim 14 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.
Claim 19
Dependent Claim
13 words
The method according to claim 14 wherein the mutagen is an electrophilic group.
Claim 20
Dependent Claim
13 words
The method according to claim 14 wherein the mutagen is an alkylating agent.
Claim 21
Dependent Claim
16 words
The method according to claim 14 wherein two or more mutagens are attached to the oligonucleotide.
Claim 22
Dependent Claim
17 words
The method according to claim 14 wherein two or more alkylating agents are attached to the oligonucleotide.
Claim 23
Dependent Claim
19 words
The method according to claim 14 wherein the mutagen is incorporated into the oligonucleotide during synthesis of the oligonucleotide.
Claim 24
Dependent Claim
20 words
The method according to claim 14 wherein the mutagen is incorporated into the oligonucleotide subsequent to synthesis of the oligonucleotide.
Claim 25
Dependent Claim
44 words
The method according to claim 7 wherein the oligonucleotide comprises at least about 26 nucleotides and wherein the oligonucleotide forms a triple-stranded complex with the target according to the rules of Watson/Crick base pairing, the method further comprising contacting the oligonucleotide with a recombinase.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-10-24 | 2004-04-24 | 2004-10-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-10-24 | 2008-04-24 | 2008-10-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-10-24 | 2012-04-24 | 2012-10-24 | $7700.00 | Window Closed / Lapsed |