EP1311709A1 - Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn - Google Patents

Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn

Info

Publication number
EP1311709A1
EP1311709A1 EP01965982A EP01965982A EP1311709A1 EP 1311709 A1 EP1311709 A1 EP 1311709A1 EP 01965982 A EP01965982 A EP 01965982A EP 01965982 A EP01965982 A EP 01965982A EP 1311709 A1 EP1311709 A1 EP 1311709A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotides
deletions
library
polynucleotide
dna
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.)
Withdrawn
Application number
EP01965982A
Other languages
German (de)
English (en)
Other versions
EP1311709A4 (fr
Inventor
Vaughn Smider
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Integrigen Inc
Original Assignee
Integrigen Inc
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 Integrigen Inc filed Critical Integrigen Inc
Publication of EP1311709A1 publication Critical patent/EP1311709A1/fr
Publication of EP1311709A4 publication Critical patent/EP1311709A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids

Definitions

  • Lietz describes a method whereby oligonucleotides with random sequences may be combined with PCR to induce insertions and deletions. Enhancement of function by this technique has not been shown, and the capacity to overmutagenize (i.e. make too many insertions or deletions per polynucleotide) is substantial in this method [Lietz, U.S. Patent # 6,251,604 (2001)]. [22] The technique most often used to evolve proteins in vitro is known as
  • compositions comprising a library of multiple (preferably more than two, more preferably more than 5, most preferably more than 10) linear polynucleotides each having a different 3' and a 5' end, but each linear polynucleotide being identical to the others if circularized are described and contemplated.
  • cleavage preferably occurs with the use of an endonuclease, preferably SI.
  • This method permits the library of addition polynucleotides to comprise any number of different polynucleotides, for example, at least 5, 10, 20 or 30 individual polynucleotides each having a random addition of nucleotides at a different position from the others.
  • the composition of multiple copies of circular polynucleotides is free of naturally-occurring homologs to the genetic element.
  • steps (a) and (b) of the method may be repeated.
  • Another option includes a process for deleting nucleotides at the point of addition in step (b).
  • FIG. 5 is a diagram illustrating an example of a method which produces short nucleotide deletions at a DNA end. Exonuclease III deletes nucleotides from the ends of a fluorescently labeled 232 bp DNA fragment in a salt dependent reaction. As salt is increased the number of deletions decreases.
  • FIG 6 is a diagram illustrating the deletion of nucleotides in the LacZ gene.
  • the plasmid pLacZi was cleaved with Cla I, treated with exonuclease III as described in FIG. 5, re-ligated, electroporated into E. coli, and plated on plates containing the colorimetric lactose analog X-Gal. Clones with either a blue or white color were picked, grown in LB, and DNA prepared. Plasmid was subjected to PCR with primers flanking the Cla I site, where one primer was fluorescently labeled.
  • the genetic element may encode a protein pharmaceutical such as a monoclonal antibody, or an enzyme used to treat a disease. Further, the genetic element may encode an enzyme important in industrial processes such as chemical manufacturing, or may be used in a product such as laundry detergent (i.e. proteases, lipases, or esterases). Further, the genetic element may have important uses in agriculture, such as to provide a means for pathogen resistance, or to allow production of novel nutrients by a plant species. Additionally, the genetic element may be used in microorganisms to produce novel products for human use, such as novel antibiotics, pigments or other small molecules. As can be seen, the modification of genetic elements in order to improve or alter their function has a myriad of applications in several diverse industries.
  • Genetic elements may be operatively linked to other genetic elements, for example a promoter may be operatively linked to a genetic element encoding a protein to allow expression of a protein in a given cell type.
  • the term "gene” and "gene of interest” refer to a polynucleotide capable of encoding a polypeptide.
  • nucleotide insertions or “nucleotide additions” means that a polynucleotide has had specific residues added to the polynucleotide chain, such that at least one of the original residues now occupies a new position in the polynucleotide when compared to the parental, wild-type, or other reference sequence.
  • library of polynucleotide sequences refers to a mixture of polynucleotides, wherein at least one of the sequences differs from at least one other sequence in the mixture by sequence composition or length, for example, where at least one position is occupied by a different nucleotide when the two sequences are compared or at least one nucleotide position is absent in one sequence when compared with the other sequence.
  • insertions at random positions in a polynucleotide of length N, it is meant that any or all of the N (in a circular polynucleotide) or N-l (in a linear polynucleotide) covalent linkages between nucleotides (i.e. phosphodiester bonds) are broken, and at least one new nucleotide (i.e. a new position) is added at the end prior to re-ligation.
  • the final length of the polynucleotide (N, or the number of positions) necessarily increases.
  • amplification means that the number of copies of a nucleic acid fragment is increased.
  • physiological conditions refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.
  • the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.
  • Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions.
  • in vitro physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-45°C.
  • aqueous conditions may be selected by the practitioner according to conventional methods.
  • buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HC1, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or nonionic detergents and/or membrane fractions and/or antifoam agents and/or scintillants.
  • the invention provides a population of polynucleotides, with members of the population differing from one another by the presence of deletions at a single random position. It is contemplated that deletions will allow removal of detrimental or unwanted functions of a genetic element. These functions might include protease sites, ion binding domains, DNA binding sequences for inhibitory transcription factors, immunogenic domains of proteins and the like.
  • the invention provides a method to generate polynucleotides wherein the polynucleotides contain insertions at more than one position.
  • One method comprises the steps of:
  • the invention provides a population of polynucleotides, with members differing from one another by a combination of deletions and insertions at a single random position. It is contemplated that this embodiment will allow for new heterologous domains to replace domains in the gene of interest. In this regard, new functions, such as ligand binding or enzymatic catalysis could be conferred upon a genetic element. Also, native function could be enhanced utilizing this embodiment.
  • the ends are linked by the intervening polynucleotide chain.
  • the re-ligation will be an intramolecular event as opposed to intermolecular, and will proceed with greater efficiency.
  • Other mechanisms to keep the ends in proximity is through a protein bridge, such as through chromatin (i.e. histones, or other DNA binding proteins), or through enzymes which couple cleavage with, rejoining, such as transposons, integrases, or topoisomerases.
  • ends could conceivably be left in proximity to one another through the linkage of opposite ends (the non-cleaved ends) to solid supports.
  • the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.
  • the templates may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).
  • viruses e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like
  • selected portions thereof e.g., coat protein, spike glycoprotein, capsid protein.
  • cosmids, phagemids, YACs, and BACs are preferred where the specific nucleic acid sequence is larger because these vectors are able to stably propagate large nucleic acid fragments.
  • Nucleotide deletions can be generated at a DNA end by a variety of means.
  • an exonuclease such as exonuclease III
  • exonuclease III can be used to remove nucleotides in a 3' to 5' direction from a DNA end.
  • the resulting DNA end contains a 5' overhang which can be removed by digestion of the DNA with a single-stranded endonuclease such as PI nuclease, SI nuclease, or mung bean nuclease.
  • Other exonucleases could also be used in the present invention.
  • Cell extracts from all organisms contain DNA repair enzymes which can act to delete nucleotides, thus unpure cell extract could conceivably be used as a source for exonuclease activity.
  • Other nucleases which may not have exonuclease activity under certain conditions may be capable of producing deletions at a DNA end under other conditions.
  • SI nuclease can produce short deletions when used at high enzyme concentrations.
  • mild denaturation of a DNA molecule such that the DNA ends become "frayed" will allow deletions to occur upon application of a single-stranded endonuclease, such as SI, PI, or mung-bean nuclease.
  • DNA ends may be rejoined by incubating the DNA ends with an enzyme like a DNA ligase which will form phosphodiester bonds between nucleotides at the DNA end.
  • ligases include E. coli DNA ligase, phage T4 DNA ligase, or human DNA ligases. These enzymes can be used under conditions well known to those skilled in the art to ligate DNA.
  • Example 2 Deletions at a site in LacZ
  • the linearized plasmid was concentrated and filtered through an ultrafree MC membrane (30 kD cutoff, Millipore, Bedford, MA), then brought to a volume of 400 ⁇ l in IX calf intestinal phosphatase buffer containing 100 U of calf intestinal phosphatase (New England Biolabs, Beverly, MA) and incubated for 45 minutes at room temperature. Plasmid was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), once with an equal volume of ether, precipitated with sodium acetate, and resuspended in water.
  • the plasmid was then incubated with exonuclease III as described in example 1, in the presence of either 100 mM, 150 mM or 200 mM NaCl for 5 minutes at 15°C in a 10 ⁇ l reaction.
  • plasmid was not incubated with exonuclease III, to test for the frequency of religation of the dephosphorylated plasmid in the absence of deletions.
  • a mix containing SI nuclease 50 U in IX SI buffer was added. This mix was further incubated at room temperature for 15 minutes. The reaction was stopped by the addition of EDTA to 0.025 M and heated to 70°C for 10 minutes.
  • the DNA was then extracted once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), once with an equal volume of ether, precipitated with sodium acetate and resuspended in 10 ⁇ l of IX ligase buffer containing 1.0 U of T4 DNA ligase (Invitrogen, Carlsbad, CA). Ligation reactions were incubated at 15°C for 12 hours. Electroporation of E. coli strain DH10B (Invitrogen, Carlsbad, CA) was accomplished with 1.0 ⁇ l of ligation mix. Cells were plated on LB agar plates containing 40 ⁇ g/ml X-Gal and 100 ⁇ g/ml ampicillin and incubated overnight at 30°C. Table 1 illustrates the results of the plating experiment.
  • Clone 5 contains a 7 basepair out of frame deletion (PCR product of 305 bases) and has a white phenotype.
  • Clone 6 has a 3 basepair deletion (PCR product of 309 bases) and has a blue phenotype. Although it may be thought that shorter deletions would lead to less severe phenotype, this experiment illustrates that this is not necessarily the case.
  • Clone 1 contains a deletion encompassing 7 amino acids but retains function whereas clones 3 and 4 contain in frame shorter deletions but do not retain function. Furthermore, this example illustrates the ability of deletional technology to search functional sequence space.
  • linearized, dephosphorylated plasmid was incubated with 1 ng of cDNA fragments in the presence of T4 DNA ligase (1.0 U) in a reaction volume of 10 ml at 15°C for 12 hours.
  • T4 DNA ligase 1.0 U
  • linearized plasmid was incubated with ligase in the absence of cDNA fragments, and cDNA fragments were incubated with ligase in the absence of linearized vector.
  • DH10B E. coli were then electroporated with 1.0 ⁇ l of each ligation mix.
  • the lac operon is a model system by which genetic elements are easily studied.
  • the enzyme ⁇ -galactosidase is encoded by the LacZ gene, but is normally only produced when lactose is present in the environment. Control of enzyme levels is accomplished at the level of transcription.
  • the lac repressor protein binds to the operator sequence upstream from the ATG start site of LacZ, and inhibits transcription by RNA polymerase. In the presence of lactose, however, the repressor is removed from the operator and transcription can proceed.
  • the mechanism of promotor activation is through the binding of lactose, the inducer, to the lac repressor and causing an allosteric change that causes its affinity for the operator to decrease dramatically.
  • LacZ transcription can be assessed by plating E. coli on the colorimetric substrate X-Gal, which causes colonies to turn blue when hydrolyzed by ⁇ -galactosidase.
  • the operator can be de- repressed by utilizing the lactose analog IPTG, which is non-hydrolizable, and strongly induces LacZ transcription by binding the lac repressor.
  • Linearized plasmid at 20 ng/ ⁇ l was incubated with 10 U exonuclease III in 66 mM Tris-Cl pH 7.4, 0.66 mM MgCl 2 buffer at 15°C for 5 minutes, followed by addition of IX SI solution containing 50 mM sodium acetate pH 4.5, 280 mM NaCl, 4.5 mM ZnSO 4 and 10 U SI nuclease, and incubation for 15 minutes at room temperature. The reaction was stopped by adding EDTA to 0.025 M, and extraction with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), once with an equal volume of ether, and precipitated with sodium acetate.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne des méthodes illustrées par la figure destinées à l'évolution dirigée, dans lesquelles des éléments génétiques sont clivés de façon aléatoire pour permettre la délétion ou l'addition de polynucléotides ou l'un et l'autre afin de créer une banque d'éléments génétiques libérés avec des additions ou des délétions. L'invention concerne également des populations de banques correspondantes. Ces processus permettent un échantillonnage significatif de l'espace de séquence nécessaire à une évolution dirigée de gènes. En outre, l'invention concerne des méthodes permettant d'effectuer de très petites délétions de nucléotides dans des éléments génétiques étudiés.
EP01965982A 2000-08-18 2001-08-17 Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn Withdrawn EP1311709A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US22647700P 2000-08-18 2000-08-18
US226477P 2000-08-18
PCT/US2001/025788 WO2002016642A1 (fr) 2000-08-18 2001-08-17 Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn

Publications (2)

Publication Number Publication Date
EP1311709A1 true EP1311709A1 (fr) 2003-05-21
EP1311709A4 EP1311709A4 (fr) 2006-08-02

Family

ID=22849054

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01965982A Withdrawn EP1311709A4 (fr) 2000-08-18 2001-08-17 Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn

Country Status (5)

Country Link
EP (1) EP1311709A4 (fr)
JP (1) JP2004507247A (fr)
AU (1) AU2001286528A1 (fr)
CA (1) CA2419961A1 (fr)
WO (1) WO2002016642A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009132220A2 (fr) * 2008-04-23 2009-10-29 Danisco Us Inc. Variants d’isoprène synthases améliorant la production microbienne d’isoprène
WO2012040571A2 (fr) * 2010-09-24 2012-03-29 Covaris, Inc. Procédé et appareil de fragmentation d'acides nucléiques

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIONDI RICHARD M ET AL: "Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 26, no. 21, 1 November 1998 (1998-11-01), pages 4946-4952, XP002187520 ISSN: 0305-1048 *
CRAMERI A ET AL: "DNA SHUFFLING OF A FAMILY OF GENES FROM DIVERSE SPECIES ACCELERATESDIRECTED EVOLUTION" NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 391, January 1998 (1998-01), pages 288-291, XP000919283 ISSN: 0028-0836 *
KIM ET AL: "Directed Evolution and Identification of Control Regions of ColE1 Plasmid Replication Origins Using Only Nucleotide Deletions" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 351, no. 4, 26 August 2005 (2005-08-26), pages 763-775, XP005004656 ISSN: 0022-2836 *
OSTERMEIER M ET AL: "A combinatorial approach to hybrid enzymes independent of DNA homology." NATURE BIOTECHNOLOGY. DEC 1999, vol. 17, no. 12, December 1999 (1999-12), pages 1205-1209, XP002179317 ISSN: 1087-0156 *
See also references of WO0216642A1 *

Also Published As

Publication number Publication date
WO2002016642A1 (fr) 2002-02-28
CA2419961A1 (fr) 2002-02-28
JP2004507247A (ja) 2004-03-11
EP1311709A4 (fr) 2006-08-02
WO2002016642A9 (fr) 2003-03-27
AU2001286528A1 (en) 2002-03-04

Similar Documents

Publication Publication Date Title
US7833759B2 (en) Method of increasing complementarity in a heteroduplex
Ostermeier et al. A combinatorial approach to hybrid enzymes independent of DNA homology
AU724698B2 (en) Recombination of polynucleotide sequences using random or defined primers
Jessberger et al. Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system
EP2009102A2 (fr) Mutagenèse aléatoire et amplification d'acide nucléique
JP6552969B2 (ja) 定方向進化のためのライブラリーの作製方法
AU2002314712A1 (en) A method of increasing complementarity in a heteroduplex polynucleotide
US7820413B2 (en) Incrementally truncated nucleic acids and methods of making same
AU2003267008B2 (en) Method for the selective combinatorial randomization of polynucleotides
EP1311709A1 (fr) Methodes et compositions pour l'evolution moleculaire dirigee utilisant une modification des extremites de l'adn
US20030036641A1 (en) Methods for homology-driven reassembly of nucleic acid sequences
US20030129709A1 (en) Method for site-directed mutagenesis of nucleic acid molecules using a single primer
WO2002081643A2 (fr) Elaboration d'echantillotheques de polynucleotides et identification d'elements de l'echantillotheque presentant les caracteristiques attendues
US20030092023A1 (en) Method of shuffling polynucleotides using templates
Pera et al. Hybrid enzymes
KR20000015984A (ko) 무작위 프라이머 또는 일정 프라이머를 이용한폴리뉴클레오티드 서열의 재조합 방법
Berta Analysis of specific cis-acting DNA sequences of the Himar1 mariner transposon
ZA200306203B (en) A method of increasing complementarity in a heteroduplex polynucleotide.
KR20000022394A (ko) 부위특이적변이도입방법
AU2002307170A1 (en) Methods for the preparation of polynucleotide librairies and identification of library members having desired characteristics
AU7257300A (en) Recombination of polynucleotide sequences using random or defined primers

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030318

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20060630

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060929