CA2473187A1 - Mismatch endonucleases and methods of use - Google Patents

Mismatch endonucleases and methods of use Download PDF

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Publication number
CA2473187A1
CA2473187A1 CA002473187A CA2473187A CA2473187A1 CA 2473187 A1 CA2473187 A1 CA 2473187A1 CA 002473187 A CA002473187 A CA 002473187A CA 2473187 A CA2473187 A CA 2473187A CA 2473187 A1 CA2473187 A1 CA 2473187A1
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Prior art keywords
endonuclease
enzyme
mismatch
polynucleotide sequence
host
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CA002473187A
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French (fr)
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CA2473187C (en
Inventor
Hal S. Padgett
Andrew A. Vaewhongs
Fakhrieh S. Vojdani
Mark L. Smith
John A. Lindbo
Wayne P. Fitzmaurice
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Novici Biotech LLC
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Priority claimed from US10/098,155 external-priority patent/US20030157495A1/en
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Publication of CA2473187C publication Critical patent/CA2473187C/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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

We describe here restriction endonucleases and their uses. Restriction endonucleases are useful in finding single nucleotide polymorphisms. They are also useful in an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences.

Claims (12)

1. An in vitro method of obtaining a peptide sequence a desired functional property, comprising:
a) preparing at least one heteroduplex polynucleotide sequence wherein said heteroduplex polynucleotide sequence has at least two non-complementary nucleotide base pairs;
b) mixing copies of the heteroduplex polynucleotide sequence with an effective amount of a mismatch endonuclease or a combination of mismatch endonucleases, a proofreading enzyme, and a ligase enzyme;
c) allowing sufficient time for non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population of polynucleotide sequence variants result.;
d) expressing polynucleotide sequence variants; and e) screening or selecting variants for the desired functional property.
2. A method according to claim 1 wherein the mismatch endonuclease is RES I endonuclease, CEL I
endonuclease, or another endonuclease of plant origin.
3. A method according to claim 1 wherein the mismatch endonuclease is a non-naturally occurring sequence variant of an endonuclease enzyme, which sequence variant has biological activity.
4. A method according to claim 1 wherein the heteroduplex polynucleotide sequence encodes endonuclease activity.
5. A method according to claim 1 or 4 wherein expressing polynucleotides variants comprises transfecting a host organism with a recombinant plasmid or a recombinant viral vector that encodes one of the polynucleotide variants.
6. A method according to claim 1 or 4 wherein expressing polynucleotides variants comprises transforming a host organism with one of the polynucleotide variants.
7. A method making a mismatch endonuclease enzyme comprising:
a) transfecting a host plant, or host animal, or host yeast or host fungus or host bacterium with a recombinant viral vector that encodes a polynucleotide sequence for a mismatch endonuclease;
b) growing the host plant, or host animal, or host yeast or host fungus or host bacterium wherein the polynucleotide sequence for a mismatch endonuclease enzyme is expressed; and c) extracting the mismatch endonuclease enzyme from the host.
8. A method according to claim 7 wherein the mismatch endonuclease enzyme is RES I or a non-naturally occurring variant thereof.
9. A method according to claim 7 wherein the mismatch endonuclease enzyme is a non-naturally occurring variant a mismatch endonuclease of plant origin.
10. A method for determining a mutation in a target sequence of a single stranded polynucleotide with reference to a non-mutated sequence of a polynucleotide that is hybridizable with the polynucleotide including said target sequence, wherein said polynucleotides are amplified, labeled with a detectable marker, hybridized to one another, subjected to the activity of an endonuclease and analyzed for the presence of said mutation, the improvement comprising the use of a non-naturally occurring mismatch endonuclease enzyme, or a mismatch endonuclease enzyme of Selaginella lepidophylla origin, the activity of said enzyme comprising:
a. detection of mismatches whether known or unknown between said hybridized polynucleotides;
b. catalytic formation of a substantially single-stranded nick at a target sequence containing a mismatch; and c. recognition of a mutation in a target polynucleotide sequence, said recognition being substantially unaffected by flanking polynucleotide sequences.
11. A method according to claim 10, further comprising d) recognition of polynucleotide loops and insertions between said hybridized polynucleotides.
12. An in vitro method of obtaining a polynucleotide sequence encoding a desired functional property, comprising:
a) preparing at least one heteroduplex polynucleotide sequence wherein said heteroduplex polynucleotide sequence has at least two non-complementary nucleotide base pairs;
b) mixing copies of the heteroduplex polynucleotide sequence with an effective amount of a mismatch endonuclease or a combination of mismatch endonucleases, a proofreading enzyme, and a lipase enzyme;
c) allowing sufficient time for non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population of polynucleotide sequence variants result; and d) screening or selecting variants for the desired functional property.
CA2473187A 2002-02-01 2003-01-31 Mismatch endonucleases and methods of use Expired - Fee Related CA2473187C (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US35372202P 2002-02-01 2002-02-01
US60/353,722 2002-02-01
US10/098,155 2002-03-14
US10/098,155 US20030157495A1 (en) 2002-02-01 2002-03-14 Nucleic acid molecules encoding CEL I endonuclease and methods of use thereof
US10/211,079 US7078211B2 (en) 2002-02-01 2002-08-01 Nucleic acid molecules encoding endonucleases and methods of use thereof
US10/211,079 2002-08-01
PCT/US2003/002958 WO2003066809A2 (en) 2002-02-01 2003-01-31 Mismatch endonucleases and methods of use

Publications (2)

Publication Number Publication Date
CA2473187A1 true CA2473187A1 (en) 2003-08-14
CA2473187C CA2473187C (en) 2012-07-31

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CA2473187A Expired - Fee Related CA2473187C (en) 2002-02-01 2003-01-31 Mismatch endonucleases and methods of use

Country Status (8)

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US (4) US7078211B2 (en)
EP (1) EP1565557B1 (en)
JP (1) JP4395632B2 (en)
AT (1) ATE366807T1 (en)
AU (1) AU2003207776B2 (en)
CA (1) CA2473187C (en)
DE (1) DE60314902T2 (en)
WO (1) WO2003066809A2 (en)

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Also Published As

Publication number Publication date
ATE366807T1 (en) 2007-08-15
DE60314902D1 (en) 2007-08-23
US7056740B2 (en) 2006-06-06
US20030148315A1 (en) 2003-08-07
US7273739B2 (en) 2007-09-25
WO2003066809A3 (en) 2005-06-30
US20030157682A1 (en) 2003-08-21
JP4395632B2 (en) 2010-01-13
US7078211B2 (en) 2006-07-18
US20080145913A1 (en) 2008-06-19
DE60314902T2 (en) 2008-04-10
AU2003207776B2 (en) 2008-12-11
WO2003066809A2 (en) 2003-08-14
CA2473187C (en) 2012-07-31
EP1565557B1 (en) 2007-07-11
EP1565557A2 (en) 2005-08-24
US20060194288A1 (en) 2006-08-31
AU2003207776A1 (en) 2003-09-02
JP2005529586A (en) 2005-10-06
EP1565557A4 (en) 2005-11-30

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