WO2008065505A3 - Simultaneous mixed site-specific mutagenesis method - Google Patents

Simultaneous mixed site-specific mutagenesis method Download PDF

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Publication number
WO2008065505A3
WO2008065505A3 PCT/IB2007/003629 IB2007003629W WO2008065505A3 WO 2008065505 A3 WO2008065505 A3 WO 2008065505A3 IB 2007003629 W IB2007003629 W IB 2007003629W WO 2008065505 A3 WO2008065505 A3 WO 2008065505A3
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WO
WIPO (PCT)
Prior art keywords
dna
mutated
template molecule
mutagenic primers
dna template
Prior art date
Application number
PCT/IB2007/003629
Other languages
French (fr)
Other versions
WO2008065505A2 (en
Inventor
Ciro Isidoro
Carlo Follo
Original Assignee
Universta Degli Studi Del Piem
Ciro Isidoro
Carlo Follo
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Universta Degli Studi Del Piem, Ciro Isidoro, Carlo Follo filed Critical Universta Degli Studi Del Piem
Publication of WO2008065505A2 publication Critical patent/WO2008065505A2/en
Publication of WO2008065505A3 publication Critical patent/WO2008065505A3/en

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Method of introducing mutations into a circular DNA template molecule comprising the steps of : i) providing two mutagenic primers; ii) annealing the mutagenic primers to the DNA template molecule; iii) synthesising two mutated DNA strands by polymeric chain reaction; iv) annealing the two mutated DNA strands obtaining a linearized mutated DNA molecule; and v) amplifying the linearized mutated DNA molecule by polymeric chain reaction; wherein the mutagenic primers present the following features : - each mutagenic primer comprises at its 3' terminal a portion having a nucleotide sequence complementary to a portion of the DNA template molecule to be mutated; the mutagenic primers anneal to the DNA template molecule back-to-back in opposite directions; and the mutagenic primers anneal to different DNA template molecule strands.
PCT/IB2007/003629 2006-11-29 2007-11-19 Simultaneous mixed site-specific mutagenesis method WO2008065505A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITTO20060848 ITTO20060848A1 (en) 2006-11-29 2006-11-29 SIMULTANEOUS MIXED PROCEDURE OF SITE-SPECIFIC DNA MUTAGENESIS
ITTO2006A000848 2006-11-29

Publications (2)

Publication Number Publication Date
WO2008065505A2 WO2008065505A2 (en) 2008-06-05
WO2008065505A3 true WO2008065505A3 (en) 2008-07-31

Family

ID=39259623

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2007/003629 WO2008065505A2 (en) 2006-11-29 2007-11-19 Simultaneous mixed site-specific mutagenesis method

Country Status (2)

Country Link
IT (1) ITTO20060848A1 (en)
WO (1) WO2008065505A2 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008067035A2 (en) * 2006-10-05 2008-06-05 Nationwide Children's Hospital Unrestricted mutagenesis and cloning methods
CN109750032A (en) * 2019-02-28 2019-05-14 尚科生物医药（上海）有限公司 A method of building gene multipoint mutation and evolution

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004072244A2 (en) * 2003-02-06 2004-08-26 Rensselaer Polytechnic Institute Polymerase-based protocols for generating chimeric oligonucleotides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004072244A2 (en) * 2003-02-06 2004-08-26 Rensselaer Polytechnic Institute Polymerase-based protocols for generating chimeric oligonucleotides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMUS: "Site-directed mutagenesis, tailored for high throughput.", INTERNET ARTICLE, 2002, XP002477881, Retrieved from the Internet <URL:http://tools.invitrogen.com/content/sfs/brochures/711_021173_Mutagens_bro.pdf> [retrieved on 20080410] *
DOMINY CLIFFORD N ET AL: "Site-directed mutagenesis by inverse PCR.", METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2003, vol. 235, 2003, pages 209 - 223, XP009099304, ISSN: 1064-3745 *
RABHI IMEN ET AL: "A novel simple and rapid PCR-based site-directed mutagenesis method", MOLECULAR BIOTECHNOLOGY, TOTOWA, NJ, US, vol. 26, no. 1, January 2004 (2004-01-01), pages 27 - 34, XP009098489, ISSN: 1073-6085 *
WEINER M P ET AL: "RAPID PCR SITE-DIRECTED MUTAGENESIS", PCR METHODS & APPLICATIONS, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 4, no. 3, 1994, pages S131 - S136, XP002043110, ISSN: 1054-9803 *

Also Published As

Publication number Publication date
ITTO20060848A1 (en) 2008-05-30
WO2008065505A2 (en) 2008-06-05

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