EP0285123A2
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EP
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A method for complete mutagenesis of nucleic acids
Assignee: Stabra AG
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Filing: 1988-03-30
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Granted: —
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Expires: 2008-03-30
Abstract
Abstract The invention relates to methods for introducing all types of base substitution mutations randomly to any nucleic acid. The methods comprise (a) preparation of the target nucleic acid to be mutagenized in the form of a single stranded template; (b) extension of an oligonucleotide hybridized to this template to generate a population of molecules, each copied from the template, and comprising molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the molecules to forms that can be amplified by molecular cloning in a suitable host; and (e) expression of the mutagenized DNA using a suitable host vector system allowing the screening for an altered gene or gene product.
Inventors
- 👤 Päivi Lehtovaara
- 👤 Jonathan Knowles
- 👤 Anu Koivula
- 👤 Jaana Bamford
- 👤 Tapani Reinikainen
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 09:19
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N9/2462; C12N9/2468; C12N9/2471; C12Y; C12Y302/00; C12Y302/01; C12Y302/01001; C12Y302/01017; C12Y302/01023
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
132 words
A method for introducing random point mutations to any nucleic acid, characterized by (a) preparation of the target nucleic acid to be mutagenized in the form of a single stranded template; (b) extension of an oligonucleotide hybridized to this template to generate a population of DNA molecules, each copied from the template, and comprising molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the DNA molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized DNA using a suitable host vector system allowing the screening for an altered gene or gene product.
Claim 2
Independent Claim
132 words
A method for introducing random point mutations to any nucleic acid, characterized by (a) preparation of the target nucleic acid to be mutagenized in the form of a single stranded template; (b) extension of an oligonucleotide hybridized to this template to generate a population of DNA molecules, each copied from the template, and comprising molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the DNA molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized DNA using a suitable host vector system allowing the screening for an altered gene or gene product.
Claim 3
Dependent Claim
16 words
The method of claim 1, characterized in that the target nucleic acid codes for a polypeptide.
Claim 4
Dependent Claim
21 words
The method of claim 1, characterized in that the target nucleic acid contains a region which functions in regulating gene expression.
Claim 5
Independent Claim
156 words
A method for preparation of a novel polypeptide including those not found in nature, characterized by (a) preparation of the target nucleic acid coding for the polypeptide to be mutagenized in the form of a single stranded template; (b) extension of an oligonucleotide hybridized to the template to generate a population of molecules, each copied from the template, and comprising molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized nucleic acids, using a suitable host vector system allowing the screening for an altered gene product; (f) screening for a clone expressing a mutant polypeptide which functions better than the unmutagenized polypeptide under the desired conditions.
Claim 6
Independent Claim
156 words
A method for preparation of a novel polypeptide including those not found in nature, characterized by (a) preparation of the target nucleic acid coding for the polypeptide to be mutagenized in the form of a single stranded template; (b) extension of an oligonucleotide hybridized to the template to generate a population of molecules, each copied from the template, and comprising molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized nucleic acids, using a suitable host vector system allowing the screening for an altered gene product; (f) screening for a clone expressing a mutant polypeptide which functions better than the unmutagenized polypeptide under the desired conditions.
Claim 7
Independent Claim
181 words
A method for altering the structure and function of nucleic acid regions which function in regulating gene expression, characterized by (a) preparation of the target nucleic acid in the form of a single stranded template, containing the regulatory region to be mutagenized and a region not to be mutagenized, coding for an easily quantitated gene product whose expression levels correlate to the structure of the regulatory region; (b) extension of an oligonucleotide hybridized to the template to generate a population of molecules, each copied from the template, and comprising of molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized nucleic acids, using a suitable host vector system allowing the screening for a clone where the mutagenized gene regulatory region functions better than the unmutagenized regulatory region under the desired conditions.
Claim 8
Independent Claim
181 words
A method for altering the structure and function of nucleic acid regions which function in regulating gene expression, characterized by (a) preparation of the target nucleic acid in the form of a single stranded template, containing the regulatory region to be mutagenized and a region not to be mutagenized, coding for an easily quantitated gene product whose expression levels correlate to the structure of the regulatory region; (b) extension of an oligonucleotide hybridized to the template to generate a population of molecules, each copied from the template, and comprising of molecules terminating at all possible nucleotide positions within a predetermined region; (c) misincorporation of nucleotides opposite to each position of the template using polymerase and the variable 3' ends generated in (b) as primers; (d) completion of the molecules generated in (c) to forms that can be amplified by molecular cloning in a suitable host; (e) expression of the mutagenized nucleic acids, using a suitable host vector system allowing the screening for a clone where the mutagenized gene regulatory region functions better than the unmutagenized regulatory region under the desired conditions.
References Cited (Backward Citations)
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