CA2485203A1 - Mutants de polymerase i d'adn sujet a l'erreur et techniques de mutagenese aleatoire ciblee en culture continue utilisant ces mutants de polymerase i d'adn sujet a l'erreur - Google Patents

Mutants de polymerase i d'adn sujet a l'erreur et techniques de mutagenese aleatoire ciblee en culture continue utilisant ces mutants de polymerase i d'adn sujet a l'erreur Download PDF

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Publication number
CA2485203A1
CA2485203A1 CA002485203A CA2485203A CA2485203A1 CA 2485203 A1 CA2485203 A1 CA 2485203A1 CA 002485203 A CA002485203 A CA 002485203A CA 2485203 A CA2485203 A CA 2485203A CA 2485203 A1 CA2485203 A1 CA 2485203A1
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CA
Canada
Prior art keywords
mutant
dna
motif
dna polymerase
pol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
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CA002485203A
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English (en)
Inventor
Manuel Camps
Lawrence A. Loeb
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University of Washington
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Individual
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Publication date
Application filed by Individual filed Critical Individual
Publication of CA2485203A1 publication Critical patent/CA2485203A1/fr
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1024—In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des formes de mutant de polymérase I présentant des mutations dans le motif A et/ou dans le motif B dans le domaine actif qui augmentent les taux d'erreur pendant la réplication. Cette invention concerne aussi des construits plasmidiques d'expression et des lignées cellulaires permettant d'exprimer ces mutants de polymérase d'ADN peu fidèles. Cette invention concerne aussi des mutants de polymérase I d'ADN peu fidèles destinés à générer des bibliothèques de gènes mutagénisés aléatoirement, lesquels peuvent être procaryotes ou eucaryotes. La mutagenèse aléatoire implique le couplage de la mutagenèse et de la sélection en culture continue pour une itération pratique, laquelle permet d'obtenir diverses plages de substitutions de paires de base généreusement réparties le long de la séquence. Parmi certains avantages, on minimise les dégradations nocives de l'ADN chromosomique et on permet une adaptation à des souches qui peuvent faire l'objet de complémentation, ce qui facilite considérablement la génération et l'identification d'enzymes aux propriétés modifiées.
CA002485203A 2002-05-31 2003-05-29 Mutants de polymerase i d'adn sujet a l'erreur et techniques de mutagenese aleatoire ciblee en culture continue utilisant ces mutants de polymerase i d'adn sujet a l'erreur Abandoned CA2485203A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US38494402P 2002-05-31 2002-05-31
US60/384,944 2002-05-31
PCT/US2003/016798 WO2003102213A2 (fr) 2002-05-31 2003-05-29 Mutants de polymerase i d'adn sujet a l'erreur et techniques de mutagenese aleatoire ciblee en culture continue utilisant ces mutants de polymerase i d'adn sujet a l'erreur

Publications (1)

Publication Number Publication Date
CA2485203A1 true CA2485203A1 (fr) 2003-12-11

Family

ID=29712109

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002485203A Abandoned CA2485203A1 (fr) 2002-05-31 2003-05-29 Mutants de polymerase i d'adn sujet a l'erreur et techniques de mutagenese aleatoire ciblee en culture continue utilisant ces mutants de polymerase i d'adn sujet a l'erreur

Country Status (5)

Country Link
EP (1) EP1504091A4 (fr)
JP (1) JP2005528114A (fr)
AU (1) AU2003237269A1 (fr)
CA (1) CA2485203A1 (fr)
WO (1) WO2003102213A2 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004005885A1 (de) * 2004-02-05 2005-10-13 Rheinische Friedrich-Wilhelms-Universität Bonn Mutierte DNA-Polymerasen mit erhöhter Fehlpaarungs-Diskriminierung
US20090269803A1 (en) * 2005-09-21 2009-10-29 Andreas Meyer In Vivo Generation of Dna, Rna, Peptide, and Protein Libraries
US8603797B2 (en) 2010-03-17 2013-12-10 Cornell University Methods and compositions for targeted mutagenesis in bacteria
JP6023173B2 (ja) * 2011-04-11 2016-11-09 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 改良された活性を有するｄｎａポリメラーゼ
CN103987843B (zh) 2011-12-08 2015-12-23 霍夫曼-拉罗奇有限公司 具有改进活性的dna聚合酶
ES2716277T3 (es) 2011-12-08 2019-06-11 Hoffmann La Roche ADN polimerasas con actividad mejorada
JP6140182B2 (ja) 2011-12-08 2017-05-31 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 改善された活性を有するｄｎａポリメラーゼ
WO2017019617A1 (fr) * 2015-07-24 2017-02-02 Manel Camps Système pour la mutagenèse continue in vivo pour faciliter une évolution dirigée
CN108130318B (zh) * 2018-02-28 2020-07-14 深圳市艾伟迪生物科技有限公司 突变型Taq DNA聚合酶、免核酸提取直接PCR扩增的试剂盒及其应用
CN112941155B (zh) * 2018-05-14 2023-07-14 北京艾克伦医疗科技有限公司 具有茎环结构的dna引物对及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69400567T2 (de) * 1994-10-17 1997-02-06 President And Fellows Of Harvard College, Cambridge, Mass. DNS Polymerase mit veränderter Nukleotid-Bindungstelle
US6306588B1 (en) * 1997-02-07 2001-10-23 Invitrogen Corporation Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof

Also Published As

Publication number Publication date
EP1504091A4 (fr) 2006-04-26
WO2003102213A3 (fr) 2004-06-17
EP1504091A2 (fr) 2005-02-09
AU2003237269A1 (en) 2003-12-19
WO2003102213A2 (fr) 2003-12-11
JP2005528114A (ja) 2005-09-22

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Legal Events

Date Code Title Description
FZDE Discontinued