US4808525A
Expired / Lapsed
US
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Site specific cleavage of DNA
Assignee: University of Georgia Research Foundation Inc
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Filing: 1987-09-11
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Granted: 1989-02-28
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Expires: 2007-09-11
Abstract
Abstract The present invention provides a method for site-specific cleavage of double stranded DNA at sequences not less than eight base pairs long, comprising methylating he DNA with a sequence-specific methylase capable of recognizing and methylating a first sequence of the DNA, thereby generating a second sequence of the DNA capable of being recognized by a site-specific endonuclease, the first and second sequences having an overlapping part thereof; the length of the combined methylase and endonuclease recognition sites being not less than eight base pairs long, and cleaving the methylated DNA by treatment with the site-specific endonuclease. This method is useful for increasing the selectivity of cutting DNA stands using restriction endonucleases, thereby permitting the isolation of large DNA fragments and the generation of unique sites in DNA fragments, e.g., cloning vectors. Also provided is a DNA vector having a terminal sequence not less than eight base pairs long which can be recognized and cut by a site-specific endonuclease.
Inventors
- 👤 Michael McClelland
- 👤 Louis G. Kessler
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 17:43
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
120 words
A method for site specific cleavage of a double-stranded DNA molecule at a recognition sequence not less than eight base pairs long, comprising: (a) contacting said double-stranded DNA molecule, which has a least two methylase recognition sequences, with at least one sequence specific methylase to produce a methylated double-stranded DNA molecule having a newly created DNA specific methylation dependent restriction endonuclease recognition sequence which partly overlaps with at least two of said methylase recognition sequences and said overlapping sequences having a combined length of not less than eight base pairs; and (b) contacting said methylated double-stranded DNA molecule with a methylation dependent DNA specific restriction endonuclease, under conditions sufficient for cleavage of the double-stranded DNA molecules by said restriction endonuclease.
Claim 2
Independent Claim
120 words
A method for site specific cleavage of a double-stranded DNA molecule at a recognition sequence not less than eight base pairs long, comprising: (a) contacting said double-stranded DNA molecule, which has a least two methylase recognition sequences, with at least one sequence specific methylase to produce a methylated double-stranded DNA molecule having a newly created DNA specific methylation dependent restriction endonuclease recognition sequence which partly overlaps with at least two of said methylase recognition sequences and said overlapping sequences having a combined length of not less than eight base pairs; and (b) contacting said methylated double-stranded DNA molecule with a methylation dependent DNA specific restriction endonuclease, under conditions sufficient for cleavage of the double-stranded DNA molecules by said restriction endonuclease.
Claim 3
Independent Claim
120 words
A method for site specific cleavage of a double-stranded DNA molecule at a recognition sequence not less than eight base pairs long, comprising: (a) contacting said double-stranded DNA molecule, which has at least one methylase recognition sequence, with at least one sequence specific methylase, to produce a methylated double-stranded DNA molecule having a newly created DNA specific methylation dependent restriction endonuclease recognition sequence which partly overlaps with at least one of said methylase recognition sequences and said overlapping sequences have a combined length of not less than 8 base pairs, and (b) contacting said methylated double-stranded DNA molecule with a methylation dependent DNA specific restriction endonuclease, under conditions sufficient for cleavage of the double-stranded DNA molecule by said restriction endonuclease.
Claim 4
Independent Claim
120 words
A method for site specific cleavage of a double-stranded DNA molecule at a recognition sequence not less than eight base pairs long, comprising: (a) contacting said double-stranded DNA molecule, which has at least one methylase recognition sequence, with at least one sequence specific methylase, to produce a methylated double-stranded DNA molecule having a newly created DNA specific methylation dependent restriction endonuclease recognition sequence which partly overlaps with at least one of said methylase recognition sequences and said overlapping sequences have a combined length of not less than 8 base pairs, and (b) contacting said methylated double-stranded DNA molecule with a methylation dependent DNA specific restriction endonuclease, under conditions sufficient for cleavage of the double-stranded DNA molecule by said restriction endonuclease.
Claim 5
Dependent Claim
16 words
The method of claim 2 wherein the endonuclease is Dpn I, Apy I or isoschizomers thereof.
Claim 6
Dependent Claim
95 words
The method of claim 2, wherein the methylases are selected from the group consisting of M. Taq I, M. Tth I, M. Cla I, M. Hph I, M. Mbo II, M. Nru I and M Xba I, M Bsp R I, M Asu I, M Nla I, M Ava II, M Sdu I, M Hgi J II, M Hgi C I, M Acy I, M Apa I, M Aac I, M Nar I, M Bst E II, M Sfi I, M Hae I, M Eco B, M Eco DXI and methylases from isoschizomer systems thereof.
Claim 7
Dependent Claim
170 words
The method of claim 2 wherein the endonuclease cleavage sequence(s) is (are) selected from the group consisting of 5' TCG m ATCG m A 3', 5' ATCG m ATCG m AT 3', 3' m AGCT m AGCT 5', 3' T m AGCT m AGCTA 5', 5' GGTG m ATCACC 3', 5' GAAG m ATCTTC 3', 3' CCACT m AGTGG 5', 3' CTTCT m AGAAG 5', 5' TCGCG m ATCGCG m A 3', 5' AGCAN 5 TGATCAN 5 TGCT 3', 3' m AGCGCT m AGCGCT 5', 3' TCGTN 5 ACTAGTN 5 ACGA 5', 5' TCTAG m ATCTAG m A 3', 5' GAATN 7 TGATCAN 7 ATTC 3', 3' m AGATCT m AGATCT 5', 3' CTTAN 7 ACTAGTN 7 TAAG 5', GGCCLGGCC, GGNCCLGGNCC, GGNNCCLGGNNCC, GGLCCLGGLCC, GXGCCCLGGGCVC, GRGCCCLGGGCYC, GGRYCCLGGYRCC, GRCGCCLGGCGYC, GGGCCCLGGGCCC, GGATCCLGGATCC, GGCGCCLGGCGCC, GGTNACCLGGTNACC, GGCCNNNNNGGCCLGGCCNNNNNGGCC, GGCCNNNNNGGCCLGGNNGGCC, and LGGCCLGGCCL; R is A or G; Y is T or C; L is A or T; N is A, C, G or T; X is A, T or G; and V is A, T or C.
Claim 8
Dependent Claim
79 words
The method of claim 2 wherein the methylase recognition sequence is selected from the group consisting of TCGA, ATCGAT, GGTGA, GAAGA, TCGCGA, TCTAGA, GGCC, GGNCC, GGNNCC, GGLCC, GXGCVC, GRGCYC, GGRYCC, GRCGYC, GGGCCC, GGATCC, GGCGCC, GGTNACC, GGCCNNNNNNNGGCC, LGGCCL, TGAN 8 TGCT, ATCAN 7 ATTC; wherein R is A or G; Y is T or C; L is A or T; N is A, C, G or T; X is A, T or G; and V is A, T or C.
Claim 9
Dependent Claim
15 words
The method of claim 2 wherein the methylation occurs on one of the DNA strands.
Claim 10
Dependent Claim
13 words
The method of claim 2 wherein the methylation occurs on both DNA strands.
Claim 11
Dependent Claim
12 words
The method of claim 2 wherein the methylation occurs on A nucleotides.
Claim 12
Dependent Claim
12 words
The method of claim 2 wherein the methylation occurs on C nucleotides.
Claim 13
Dependent Claim
15 words
The method of claim 1 wherein the cleavage occurs at an 8 base pair sequence.
Claim 14
Dependent Claim
12 words
The method of claim 11 wherein the 8 base-pair sequence is TCGATCGA.
Claim 15
Dependent Claim
15 words
The method of claim 1 wherein the cleavage occurs at a 10 base pair sequence.
Claim 16
Dependent Claim
12 words
The method of claim 13 wherein the 10 base-pair sequence is ATCGATCGAT.
Claim 17
Dependent Claim
176 words
DNA vector having a double stranded DNA sequence of not less than 8 base pairs long selected from the group consisting of TCGATCGA, ATCGATCGAT, 5' TCG m ATCG m A 3', 5' ATCG m ATCG m AT 3', 3' m AGCT m AGCT 5', 3' T m AGCT m AGCTA 5', 5' GGTG m ATCACC 3', 5' GAAG m ATCTTC 3', 3' CCACT m AGTGG 5', 3' CTTCT m AGAAG 5', 5' TCGCG m ATCGCG m A 3', 5' AGCAN 5 TGATCAN 5 TGCT 3', 3' m AGCGCT m AGCGCT 5', 3' TCGTN 5 ACTAGTN 5 ACGA 5', 5' TCTAG m ATCTAG m A 3', 5' GAATN 7 TGATCAN 7 ATTC 3', 3' m AGATCT m AGATCT 5', 3' CTTAN 7 ACTAGTN 7 TAAG 5', GGCCLGGCC, GGNCCLGGNCC, GGNNCCLGGNNCC, GGLCCLGGLCC, GXGCCCLGGGCVC, GRGCCCLGGGCYC, GGRYCCLGGYRCC, GRCGCCLGGCGYC, GGGCCCLGGGCCC, GGATCCLGGATCC, GGCGCCLGGCGCC, GGTNACCLGGTNACC, GGCCNNNNNGGCCLGGCCNNNNNGGCC, GGCCNNNNNGGCCLGGNNGGCC, and LGGCCLGGCCL; R is A or G; Y is T or C; L is A or T; N is A, C, G or T; X is A, T or G; and V is A, T or C.
Claim 18
Dependent Claim
176 words
DNA vector having a double stranded DNA sequence of not less than 8 base pairs long selected from the group consisting of TCGATCGA, ATCGATCGAT, 5' TCG m ATCG m A 3', 5' ATCG m ATCG m AT 3', 3' m AGCT m AGCT 5', 3' T m AGCT m AGCTA 5', 5' GGTG m ATCACC 3', 5' GAAG m ATCTTC 3', 3' CCACT m AGTGG 5', 3' CTTCT m AGAAG 5', 5' TCGCG m ATCGCG m A 3', 5' AGCAN 5 TGATCAN 5 TGCT 3', 3' m AGCGCT m AGCGCT 5', 3' TCGTN 5 ACTAGTN 5 ACGA 5', 5' TCTAG m ATCTAG m A 3', 5' GAATN 7 TGATCAN 7 ATTC 3', 3' m AGATCT m AGATCT 5', 3' CTTAN 7 ACTAGTN 7 TAAG 5', GGCCLGGCC, GGNCCLGGNCC, GGNNCCLGGNNCC, GGLCCLGGLCC, GXGCCCLGGGCVC, GRGCCCLGGGCYC, GGRYCCLGGYRCC, GRCGCCLGGCGYC, GGGCCCLGGGCCC, GGATCCLGGATCC, GGCGCCLGGCGCC, GGTNACCLGGTNACC, GGCCNNNNNGGCCLGGCCNNNNNGGCC, GGCCNNNNNGGCCLGGNNGGCC, and LGGCCLGGCCL; R is A or G; Y is T or C; L is A or T; N is A, C, G or T; X is A, T or G; and V is A, T or C.
Claim 19
Dependent Claim
13 words
The DNA vector of claim 15 containing a self-replicating sequence of viral origin.
Claim 20
Dependent Claim
13 words
The DNA vector of claim 15 containing a self-replicating sequence of plasmid origin.
Claim 21
Dependent Claim
9 words
The DNA vector of claim 15 of eukaryotic origin.
Claim 22
Dependent Claim
9 words
The DNA vector of claim 15 of prokaryotic origin.
Claim 23
Dependent Claim
19 words
The DNA vector of claim 15 capable of replicating in an eukaryotic organism, a prokaryotic organism or in both.
Claim 24
Dependent Claim
19 words
A double stranded DNA comprising: (a) the vector of claim 15, and (b) a foreign DNA fragment cloned therein.
References Cited (Backward Citations)
US4419446A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-02-28 | 1992-08-29 | 1993-02-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-02-28 | 1996-08-29 | 1997-02-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-02-28 | 2000-08-29 | 2001-02-28 | $7700.00 | Window Closed / Lapsed |