US5776744A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods and compositions for effecting homologous recombination
Assignee: Yale University
•
Filing: 1995-06-07
•
Granted: 1998-07-07
•
Expires: 2015-06-07
Abstract
Abstract Methods, compositions and kits for effecting homologous recombination are described. In one aspect, the method utilizes two introduced DNAs: (1) a mutagen-linked single-stranded oligonucleotide capable of specifically binding to double-stranded DNA to form a triple-stranded helix, and (2) a donor DNA fragment capable of undergoing homologous recombination with DNA targeted by the oligonucleotide.
Inventors
- 👤 Peter M. Glazer
- 👤 L. Michael Lin
- 👤 Jay George
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 19:48
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12N15/87; C12N15/90; C12N15/902; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6839; C12N2310/00; C12N2310/10; C12N2310/15; C12N2310/30; C12N2310/35; C12N2310/351; C12N2310/3511; C12N2310/3515; C12N2310/50; C12N2310/53
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
201 words
A method for effecting homologous recombination between a native nucleic acid segment in a cell and a donor nucleic acid segment introduced into the cell, which comprises: a) introducing into a cell: i) an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of a native nucleic acid segment in the vicinity of a target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the native nucleic acid, and ii) a donor nucleic acid which comprises a nucleic acid sequence sufficiently homologous to the native nucleic acid segment such that the donor sequence is capable of undergoing homologous recombination with the native sequence at the target region; b) allowing the oligonucleotide to bind to the native nucleic acid segment to form a triple stranded nucleic acid, thereby inducing homologous recombination at the native nucleic acid segment target region; and c) allowing homologous recombination to occur between the native and donor nucleic acid segments wherein the oligonucleotide third strand has a dissociation constant for the binding region of less than or equal to about 10 -7 M.
Claim 2
Independent Claim
201 words
A method for effecting homologous recombination between a native nucleic acid segment in a cell and a donor nucleic acid segment introduced into the cell, which comprises: a) introducing into a cell: i) an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of a native nucleic acid segment in the vicinity of a target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the native nucleic acid, and ii) a donor nucleic acid which comprises a nucleic acid sequence sufficiently homologous to the native nucleic acid segment such that the donor sequence is capable of undergoing homologous recombination with the native sequence at the target region; b) allowing the oligonucleotide to bind to the native nucleic acid segment to form a triple stranded nucleic acid, thereby inducing homologous recombination at the native nucleic acid segment target region; and c) allowing homologous recombination to occur between the native and donor nucleic acid segments wherein the oligonucleotide third strand has a dissociation constant for the binding region of less than or equal to about 10 -7 M.
Claim 3
Dependent Claim
20 words
The method of claim 1, wherein the oligonucleotide third strand is from about 7 to about 50 nucleotides in length.
Claim 4
Dependent Claim
20 words
The method of claim 2, wherein the oligonucleotide third strand is from about 10 to about 30 nucleotides in length.
Claim 5
Dependent Claim
17 words
The method of claim 1, wherein the oligonucleotide third strand contains an at least partially artificial backbone.
Claim 6
Dependent Claim
27 words
The method of claim 1, wherein the oligonucleotide third strand contains a backbone selected from the group consisting of phosphodiester, phosphorothioate, methyl phosphonate, peptide, and mixtures thereof.
Claim 7
Dependent Claim
10 words
The method of claim 5, wherein the backbone is phosphodiester.
Claim 8
Dependent Claim
18 words
The method of claim 1, wherein the oligonucleotide third strand is modified with one or more protective groups.
Claim 9
Dependent Claim
24 words
The method of claim 7, wherein the 3' and 5' ends of the oligonucleotide third strand are modified with one or more protective groups.
Claim 10
Dependent Claim
21 words
The method of claim 7, wherein the protective group is selected from the group consisting of alkyl amines, acridine and cholesterol.
Claim 11
Dependent Claim
12 words
The method of claim 1, wherein the oligonucleotide third strand is circularized.
Claim 12
Dependent Claim
16 words
The method of claim 1, wherein the oligonucleotide third strand contains at least one modified sugar.
Claim 13
Dependent Claim
16 words
The method of claim 1, wherein the oligonucleotide third strand comprises at least one synthetic base.
Claim 14
Dependent Claim
19 words
The method of claim 1, wherein the dissociation constant is less than or equal to about 2×10 -8 M.
Claim 15
Dependent Claim
20 words
The method of claim 1, wherein the oligonucleotide third strand has linked thereto a moiety which induces the homologous recombination.
Claim 16
Dependent Claim
14 words
The method of claim 14, wherein the moiety is linked to the oligonucleotide directly.
Claim 17
Dependent Claim
16 words
The method of claim 14, wherein the moiety is linked to the oligonucleotide through a linker.
Claim 18
Dependent Claim
34 words
The method of claim 14, wherein the moiety is selected from the group consisting of acridine, psoralen, a substituted psoralen, hydroxymethylpsoralen, mitomycin C, 1-nitrosopyrene, a nuclease, a restriction enzyme, a radionuclide, boron, and iodine.
Claim 19
Dependent Claim
13 words
The method of claim 1, wherein the donor nucleic acid is double stranded.
Claim 20
Dependent Claim
13 words
The method of claim 1, wherein the donor nucleic acid is single stranded.
Claim 21
Dependent Claim
16 words
The method of claim 1, wherein the donor nucleic acid comprises two substantially complementary single strands.
Claim 22
Dependent Claim
18 words
The method of claim 1, wherein the donor nucleic acid is substantially homologous with the native nucleic acid.
Claim 23
Dependent Claim
33 words
The method of claim 21, wherein the donor nucleic acid is substantially homologous with the native nucleic acid in a region of about 20 bases at each end of the donor nucleic acid.
Claim 24
Dependent Claim
18 words
The method of claim 1, wherein the donor nucleic acid is at least about 40 bases in length.
Claim 25
Dependent Claim
20 words
The method of claim 23, wherein the donor nucleic acid is between about 40 and about 40,000 bases in length.
Claim 26
Dependent Claim
22 words
The method of claim 1, wherein the donor nucleic acid is introduced into the cell in the form of a packaging system.
Claim 27
Dependent Claim
25 words
The method of claim 25, wherein the packaging system is selected from the group consisting of a DNA virus, an RNA virus, and a liposome.
Claim 28
Dependent Claim
20 words
The method of claim 1, wherein the native nucleic acid contains a mutation that is corrected by the homologous recombination.
Claim 29
Dependent Claim
24 words
The method of claim 27, wherein the mutation is selected from the group consisting of base changes, deletions, insertions, nucleotide repeats, and combinations thereof.
Claim 30
Dependent Claim
18 words
The method of claim 1, wherein the homologous recombination causes an alteration in the native nucleic acid sequence.
Claim 31
Dependent Claim
47 words
The method of claim 29, wherein the alteration is an addition of a segment selected from the group consisting of a gene, a part of a gene, a gene control region, an intron, a splice junction, a transposable element, a site specific recombination sequence, and combinations thereof.
Claim 32
Dependent Claim
12 words
The method of claim 1, wherein the native nucleic acid is chromosomal.
Claim 33
Dependent Claim
12 words
The method of claim 1, wherein the native nucleic acid is extrachromosomal.
Claim 34
Dependent Claim
24 words
The method of claim 32, wherein the native nucleic acid is selected from the group consisting of mitochondrial, episomal, a plasmid and a chloroplast.
Claim 35
Independent Claim
216 words
A method for effecting homologous recombination between a first nucleic acid segment in a cell and a donor nucleic acid segment introduced into the cell, which comprises: a) contacting a donor nucleic acid segment with an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of the donor nucleic acid segment in the vicinity of a target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the donor nucleic acid, and said donor having a sequence sufficiently homologous to a first nucleic acid segment such that the donor sequence will undergo homologous recombination with the first sequence at the target region; b) allowing the oligonucleotide to bind to the donor nucleic acid segment to form a triple stranded nucleic acid, thereby treating the donor nucleic acid segment to make it capable of inducing homologous recombination at the donor nucleic acid segment target region; c) introducing into a cell the treated donor nucleic acid; and d) allowing homologous recombination to occur between the first and donor nucleic acid segments wherein the oligonucleotide third strand has a dissociation constant for the binding region of less than or equal to about 10 -7 M.
Claim 36
Independent Claim
216 words
A method for effecting homologous recombination between a first nucleic acid segment in a cell and a donor nucleic acid segment introduced into the cell, which comprises: a) contacting a donor nucleic acid segment with an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of the donor nucleic acid segment in the vicinity of a target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the donor nucleic acid, and said donor having a sequence sufficiently homologous to a first nucleic acid segment such that the donor sequence will undergo homologous recombination with the first sequence at the target region; b) allowing the oligonucleotide to bind to the donor nucleic acid segment to form a triple stranded nucleic acid, thereby treating the donor nucleic acid segment to make it capable of inducing homologous recombination at the donor nucleic acid segment target region; c) introducing into a cell the treated donor nucleic acid; and d) allowing homologous recombination to occur between the first and donor nucleic acid segments wherein the oligonucleotide third strand has a dissociation constant for the binding region of less than or equal to about 10 -7 M.
References Cited (Backward Citations)
WO1992008791A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-07-06 | 2002-01-05 | 2002-07-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-07-06 | 2006-01-05 | 2006-07-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-07-06 | 2010-01-05 | 2010-07-07 | $7700.00 | Window Closed / Lapsed |