EP0193413A2
Expired / Lapsed
EP
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Cloning restriction and modification genes
Assignee: New England Biolabs Inc
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Filing: 1986-03-03
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Granted: —
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Expires: 2006-03-03
Abstract
Abstract A method of cloning a restriction gene comprises forming a library containing DNA coding for the restriction gene, isolating clones which contain the modification gene and screening the clones containing the modification gene for the presence of the restriction gene. Clones containing the restriction gene can be cultured to provide restriction and/or corresponding modification enzymes.
Inventors
- 👤 Geoffrey Graham Wilson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 21:43
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/16; C12N9/22; C12N9/10; C12N9/1003; C12N9/1007
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
40 words
A method of cloning a restriction gene comprising forming a library containing DNA coding for the restriction gene, isolating clones which contain the modification gene and screening the clones containing the modification gene for the presence of the restriction gene.
Claim 2
Independent Claim
40 words
A method of cloning a restriction gene comprising forming a library containing DNA coding for the restriction gene, isolating clones which contain the modification gene and screening the clones containing the modification gene for the presence of the restriction gene.
Claim 3
Independent Claim
76 words
The method of claim I, wherein the library is formed by the steps of: (a) purifying DNA from a source containing the restriction gene; (b) partially digesting the purified DNA to form DNA fragments; (c) ligating the fragments into a cloning vector; (d) transforming a host cell with the cloning vector of step (c) to form a primary cell library; and (e) purifying recombinant vectors from the primary cell library to form a primary vector library.
Claim 4
Dependent Claim
17 words
The method of claim 2, wherein the source of DNA is a bacteria containing the restriction gene.
Claim 5
Dependent Claim
16 words
The method of claim 2, wherein the cloning vector is a plasmid or viral DNA molecule.
Claim 6
Dependent Claim
10 words
The method of claim 4, wherein the plasmid is pBR322.
Claim 7
Dependent Claim
20 words
The method of claim 4, wherein the host cell is a strain of E, coli which is hsdR - .
Claim 8
Dependent Claim
48 words
The method of claim 1, wherein the clone containing the modification gene is isolated by digesting to completion the library with restriction enzyme corresponding to the modification gene to form a digestion pool, retransforming the digestion pool in a host cell and selecting clones containing the modification gene.
Claim 9
Dependent Claim
49 words
The method of claim 2, wherein the clone containing the modification gene is isolated by digesting to completion the vector library with restriction enzyme corresponding to the modification gene to form a digestion pool, retransforming the digestion pool in a host cell and selecting clones containing the modification gene.
Claim 10
Dependent Claim
17 words
The method of claim 8, wherein digestion is supplemented by the additicn of an exonuclease or phosphatase.
Claim 11
Dependent Claim
54 words
The method of claim 8, including the additional steps of : (a) forming a further vector library from the digestion pool; (b) redigesting the vector library of step (a) with the restriction enzyme to form a digestion pool; and (c) selecting clones containing the modification gene prepared from the digestion pool of step (b).
Claim 12
Dependent Claim
20 words
The method of claim 10, wherein steps (a) and (b) are repeated to facilitate selection in accordance with step (c).
Claim 13
Dependent Claim
11 words
A clone produced in accordance with the method of claim 1.
Claim 14
Dependent Claim
13 words
The clone of claim 12, wherein the restriction gene codes for Taq I.
Claim 15
Dependent Claim
13 words
The clone of claim 12, wherein the restriction gene codes for Hae II.
Claim 16
Independent Claim
114 words
A method for producing a restriction enzyme and/or its corresponding modification enzyme comprising the steps: (a) purifying DNA from a source containing the restriction gene; (b) partially digesting the purified DNA to form DNA fragments; (c) ligating the fragments into a cloning vector; (d) transforming a host cell with the cloning vector of step (c) to form a library; (e) screening the library for clones containing a modification gene corresponding to the restriction gene by digesting to completion with the.restriction enzyme; (f) further screening the clones of step (e) for the presence of the restriction gene; (g) culturing the clones of step (f); and (h) recovering the restriction and/or modification enzymes from the culture.
Claim 17
Independent Claim
114 words
A method for producing a restriction enzyme and/or its corresponding modification enzyme comprising the steps: (a) purifying DNA from a source containing the restriction gene; (b) partially digesting the purified DNA to form DNA fragments; (c) ligating the fragments into a cloning vector; (d) transforming a host cell with the cloning vector of step (c) to form a library; (e) screening the library for clones containing a modification gene corresponding to the restriction gene by digesting to completion with the.restriction enzyme; (f) further screening the clones of step (e) for the presence of the restriction gene; (g) culturing the clones of step (f); and (h) recovering the restriction and/or modification enzymes from the culture.
Claim 18
Dependent Claim
17 words
The method of claim 15, wherein the source of DNA is a bacteria containing the restriction gene.
Claim 19
Dependent Claim
12 words
The method of claim 15, wherein the cloning vector is a plasmid.
Claim 20
Dependent Claim
10 words
The method of claim 17, wherein the plasmid is pBR322.
Claim 21
Dependent Claim
18 words
The method of claim 15, wherein the host cell is a strain of E. coli which is hsdR-.
Claim 22
Dependent Claim
11 words
Restriction enzyme produced in accordance with the method of claim 19.
Claim 23
Dependent Claim
21 words
The method of claim 15, wherein the restriction enzyme produced is Hae II and the source of DNA is Haemophilus aegyptius.
Claim 24
Dependent Claim
11 words
Hae II produced in accordance with the method of claim 21.
Claim 25
Dependent Claim
11 words
M.Hae II produced in accordance with the method of claim 21.
Claim 26
Dependent Claim
21 words
The method of claim 15, wherein the restriction enzyme produced is Taq I and the source of DNA is Thermus aquaticus.
Claim 27
Dependent Claim
11 words
Taq I produced in accordance with the method of claim 24.
Claim 28
Dependent Claim
11 words
M.Taq I produced in accordance with the method of claim 24.
Claim 29
Independent Claim
23 words
A method of cloning a cytosine-type modification methylase gene comprising constructing the clone and propagating it in an Rgl-deficient strain of E, coli.
Claim 30
Independent Claim
23 words
A method of cloning a cytosine-type modification methylase gene comprising constructing the clone and propagating it in an Rgl-deficient strain of E, coli.
Claim 31
Dependent Claim
13 words
The method of claim 27, wherein the strain of E.coli is Rgl A-deficient.
Claim 32
Dependent Claim
14 words
The method of claim 27, wherein the strain of E. coli is Rgl B-deficient.
Claim 33
Dependent Claim
17 words
The method of claim 27, wherein the strain of E. coli is Rgl A- and Rgl B-deficient.
Claim 34
Dependent Claim
20 words
The method of claim 27, wherein the clone also codes for the restriction endonuclease corresponding to the cytosine-type modification methylase.
References Cited (Backward Citations)
No citation records indexed.
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