WO1994018333A1 - METHODE DE CLONAGE D'ADN A L'AIDE DU VECTEUR Cre-Lox DANS DES CONDITIONS D'ENCOMBREMENT MACROMOLECULAIRE - Google Patents

METHODE DE CLONAGE D'ADN A L'AIDE DU VECTEUR Cre-Lox DANS DES CONDITIONS D'ENCOMBREMENT MACROMOLECULAIRE Download PDF

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Publication number
WO1994018333A1
WO1994018333A1 PCT/GB1994/000272 GB9400272W WO9418333A1 WO 1994018333 A1 WO1994018333 A1 WO 1994018333A1 GB 9400272 W GB9400272 W GB 9400272W WO 9418333 A1 WO9418333 A1 WO 9418333A1
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dna
vector
cre
cloning
linear
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PCT/GB1994/000272
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English (en)
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Alan Christopher Boyd
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Medical Research Council
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C12N15/68—Stabilisation of the vector
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • This invention relates to a rapid method of cloning a DNA molecule, particularly a blunt-ended DNA molecule, and to a novel plasmid of use in performing the method.
  • This application concerns a similar but more straightforward two-stage technique in which the second stage restriction/ligation steps of the published method (6) are replaced by a single, rapid site-specific recombination mediated by the lox/Cre system of bacteriophage Pl(7).
  • Cre is a recombinase which acts specifically on the 34bp lox sequence: its natural function is to circularize PI genomes which when injected into cells are linear lOOkb molecules flanked by lox sites in direct repeat (8).
  • Cre is not fastidious and works efficiently in vitro under conditions similar to those used for restriction enzyme digestion (9).]
  • the principle of the second stage has been outlined previously (10) and proposed as a means of cloning large fragments into plasmids.
  • the addition here of the highly efficient ligation step allows this principle to be exploited in a more general technique for plasmid cloning.
  • the whole process, designated 'turbo cloning' is quick (1 - 2h), simple and efficient.
  • the present invention provides a method of cloning a DNA molecule, comprising: a) ligating a linear insert DNA molecule to linear vector DNA molecules, which vector molecules contain a single lox site, under conditions of macromolecular crowding so as to form at least some suitably ligated DNA in which a linear insert DNA molecule is joined at each end to a respective molecule of linear vector DNA wherein the two linear vector DNA molecules immediately adjacent to the insert DNA have the relationship of being direct repeats; b) bringing the products of the ligation reaction, no longer under conditions of macromolecular crowding, into contact with Cre protein so as to cause circularisation of the suitably ligated DNA; and c) dissociating the Cre protein from the DNA.
  • Step b) may be achieved intracellularly by introducing the DNA into a cell which is capable of expressing Cre, preferably using a cell which is capable of inducible expression of Cre.
  • step b) may be achieved extracellularly, in which case the method further comprises as step ) introducing the DNA into a suitable host.
  • the Cre protein is conveniently dissociated from the DNA by heating.
  • Conditions of macromolecular crowding can be achieved by the presence of 15% (w/v) polyethylene glycol 6000.
  • the conditions of macromolecular crowding are conveniently removed by dilution of the reaction mixture.
  • the vector molecule is preferably p91ox5.
  • the vector has preferably been dephosphorylated prior to ligation.
  • the linear insert DNA may have blunt ends.
  • the present invention provides the plasmid p91ox5 as herein defined.
  • Figure 1 shows results obtained on an agarose gel
  • FIG. 2 illustrates schematically the method of the present invention
  • Figure 3 shows further results obtained on an agarose gel.
  • DNAs were prepared by standard methods ( 1 ) and restriction enzymes used according to the supplier's (Boehringer) recommendations.
  • a Sail cohesive ended duplex containing lox was made by annealing the 40-mers B16
  • stage one the vector and fragment DNAs (in water or TE: 10mM Tris.HCKpH 8.0), 1mM EDTA) are combined in a ligation buffer similar to that described (4) (final concentrations: 50mM Tris.HCl (pH 8.0), 0.5mM ATP, 0.5mM dithioerythritol (DTE), 5mM MgCl 2 ) and an appropriate volume of PEG 6000 (BDH) added from a filter-sterilized 40% (w/v) stock solution to give a final concentration of 15%.
  • 0.3 to 0.5 units of T4 DNA ligase (Boehringer) is added giving a total reaction volume of 10 - 25ul. The reaction is mixed carefully and thoroughly by pumping through a micropipettor tip before incubation at room temperature for 10 - 30 min. The reaction is terminated by heat inactivation of ligase at 75° for 10 - 15 min.
  • the second stage is initiated by adding four volumes of Cre buffer (1OmM Tris.HCl(pH7.5), 1OmM MgCl 2 , 50mM NaCl, 1mM DTE) containing 0.15ug of Cre protein (NEN).
  • Cre buffer (1OmM Tris.HCl(pH7.5), 1OmM MgCl 2 , 50mM NaCl, 1mM DTE) containing 0.15ug of Cre protein (NEN).
  • Cre buffer 1OmM Tris.HCl(pH7.5), 1OmM MgCl 2 , 50mM NaCl, 1mM DTE
  • Cre protein N- 30 min before heat inactivation as before.
  • Desalting was achieved by 15min drop dialyses of small aliquots of reaction mix on nitrocellulose filters (Millipore type VM, 0.05u pore size) floating on water (15). 1-2ul of the dialysate was electroporated into 30 - 35ul of electrocompetent cells (Bio-Rad Gene Pulser: 2.5kV, 25uF, 200 ohm). Subsequent steps were as described (14), except that all media used were L broth based.
  • DNA fragments were removed by Geneclean (Bio 101) extraction and the DNA eluted into water.
  • the blunt ended fragments were them phosphorylated by polynucleotide kinase (Boehringer) as described (18).
  • the enzyme was heat inactivated and a 15 min drop dialysis step (see above) was used to remove salts and the DNA concentration estimated by running an aliquot on a gel.
  • the first stage is common to both methods.
  • a plasmid vector containing a lox site must be used: linearized, blunt-ended vector (p91ox5) and fragment DNAs are ligated in 15% PEG at room temperature.
  • blunt end ligation is almost as efficient as cohesive end ligation (4), and essentially all phosphorylated termini are joined in a short time (less than 1h) by moderate amounts of T4 DNA ligase.
  • T4 DNA ligase To maximise recovery of distinct recombinants, there must be a large molar excess of vector so that most insert DNA fragments become flanked by vector molecules.
  • stage 2 the reaction mix is diluted to abolish macromolecular crowding and Cre protein added.
  • the 50% of hybrids with lox in direct repeat will be productively resolved to the desired circular recombinants (plus vector monomers).
  • the other hybrids will undergo unproductive rounds of inversion between lox sites and remain linear.
  • turbo cloning is subject to a theoretical maximum efficiency of 50%.
  • Cre works in the opposite direction of integrating circles into linears to regenerate linear hybrids, the equilibrium is strongly in favour of circle formation (19).)
  • stage 2 is complete and the DNA can be introduced into cells.
  • a plasmid containing a selectable marker was chosen as the source of insert fragment for convenient identification of recombinants.
  • the 4.3kb plasmid pACBl04 replicates via the lambda dv replico ⁇ and carries the CAT gene encoding resistance to chloramphenicol (21): it was linearized at the unique EcoRV site within the 0 gene generating a blunt-ended fragmemt.
  • Plasmids from 36 clones were examined both by Alu-PCR and restriction analysis: six had Alu-PCR fragments (ranging in size from 0.2 to 2kb) inserted (Fig. 3), the rest, reconstituted vectors, are presumed to have arisen from incomplete Smal digestion or dephosphorylation of p91ox5. The yield of recombinants is therefore about 2 x 10° per ug fragment DNA. Given that the Alu-PCR fragments had to be blunt ended and phosphorylated prior to cloning, this yeld of recombinants is gratifyingly high. Restriction mapping of the recombinants (data not shown) proved them to be unrelated. Thus, turbo cloning permits the efficient recovery of PCR products with minimal preprocessing and low selectivity.
  • turbo cloning produces recombinants from blunt-ended fragments at levels comparable to those obtained previously (6).
  • the method may also be used for the cloning of cohesive-ended fragments, but the increase in efficiency is not so great. Nevertheless, as in the 10% PEG method (11), the saving in time may make it an attractive method to use for both kinds of cloning).
  • the ease with which PCR products can be cloned is particularly notable, since many other methods rely on including restriction sites at the 5 1 end of primers to permit cohesive-end cloning of products. With turbo cloning, there is no need for such considerations in primer design.
  • the disadvantages of (6) are (i) the time-consuming restriction and ligation steps of stage 2 and (ii) the potential problem of cloning inserts which contain sites for the second stage restriction enzyme.
  • the use of a rare cutter like Notl may substantially reduce (ii): turbo cloning does not have this problem because lox sites, being 34bp long, are unlikely to occur at all in most genomes.
  • the new method unlike others, requires specialized plasmid vectors carrying lox.
  • a more versatile plasmid than p91ox5 derived from BlueScri'oe (22) and containing lox outwith the alpha-complementing lacZ' gene fragment (thus allowing the blue/white screen for recombinants) has now also been constructed for this purpose .
  • turbo cloning could usefully be applied (as with (6)) to the generation of cDNA plasmid libraries.
  • Blunt-ended cDNAs could be cloned directly and efficiently, with many fewer steps. However, this would require the use of maximally electro ⁇ ompetent cells to compete with the efficiencies obtained with lambda expression vectors (1).
  • turbo cloning and similar methods may be the recovery of DNA from scarce archive material such as paraffin blocks, forensic samples and ancient biological specimens.
  • the fraction of DNA surviving in an intact state in this material is usually tiny (24): because turbo cloning maximises the recovery of small amounts of blunt-ended DNA, and requires minimal preprocessing (making ends flush with T4 DNA polymerase may be all that is required), it could be more effective than existing approaches (25,26).
  • PCR methods for examining such material though extremely sensitive, suffer from size and sequence selectivity and can give misleading results due to strand switching and misincorporation of bases during amplification.
  • the Cre step of turbo cloning may be achieved in vivo.
  • 80ng of Smal-cleaved p91ox5 was ligated to 100ng Smal- cleaved pACBl04 in the presence of 15% PEG 6000.
  • Some recombinant plasmids (after transfer to JM83 for ease of purification) were analysed by restriction digestion and shown to contain the vector and insert fragments, though complex chimaeras with multiple components were also observed. This is to be expected if, as in the experiment described here, the molar excess of vector over fragment is low.
  • turbo cloning could be simplified further by carrying out the Cre circularisation step in vivo rather than in vitro, for example by use of a transiently induced Cre gene to confine Cre expression to the time at which linear concatemeric DNA from the PEG ligation step was transformed into cells.
  • this could more simply be achieved with the use of a suitable host/vector system in which the host strain would contain a plasmid expressing Cre constitutively.
  • the incoming turbo cloning vector would be designed to switch off replication of the resident plasmid, which would then rapidly be lost during colony formation.
  • the components of the system would be as follows:
  • Host strain an E. coli strain carrying a plasmid (with a different origin of replication and antibiotic resistance determinant from the turbo cloning vector) expressing the Cre protein.
  • this could be the lambda dv-based chloramphenicol resistance encoding plasmid pACBl04 (Boyd et al. Mol. Gen. Genet., (1989), 217, 488- 498) with the Cre gene cloned into the polylinker under control of the lac promoter. It may be desirable also to use a strain lacking RecBCD nuclease activity to minimise degradation of incoming linear DNA prior to Cre-mediated circularisation (Sauer & Henderson, Gene, .(1988), 70,331-341).
  • Vector a plasmid carrying a lox site and a trans- acting incompatibility determinant specific for the Cre-expressing plasmid in the host strain.
  • a suitable determinant would be the lambda cT or cro gene under control of a strong constitutive promoter.
  • Such a vector could be made by cloning this promoter-cl/cro cassette into an existing turbo cloning vector such as p91ox5 . described in the published work (Boyd, Nucl.Acids Res. (1993), 21, 817-821).
  • vector and insert DNA ligated in the presence of 15% PEG 6000 as in the standard protocol would be transformed directly into competent cells of the host strain. Selection would then be applied for the incoming plasmid. Cre protein already in the cells produced by the resident plasmid would carry out the necessary circularisation of incoming DNA. The incompatibility determination on the incoming plasmid would then be expressed and abolish replication of the resident plasmid. By the time a colony was formed, no trace of the Cre expression plasmid would remain to complicate analysis of recombinants.
  • V:F ratios were achieved by ligating constant amounts of (30ng) of vector (using 0.3 units T4 DNA ligase -
  • Cre reaction was carried out in a volume of 60ul for 30 min at 30°.
  • 100ng of Smal or EcoRI-cut dephosphorylated vector (p91ox5) were ligated to 16ng EcoRV- or EcoRI-cut fragment (pACBl04) in two turbo cloning reactions otherwise carried out as in footnote c of Table 1.
  • 26ng uncut p91ox5Cm were processed similarly and gave rise to 3.2 x 10' colonies per ug fragment equivalent.
  • FIG. 1 Alu-PCR products of 32.13 DNA. DNA was amplified as in Materials and Methods, and 20ul aliquots of three independent 50ul reactions were run on a 1.8% agarose gel (lanes 2 - 4). Lane 1 contains size markers (1kb ladder, BRL: sizes shown in kb).
  • FIG. 1 Overview of turbo cloning, (a) Stage 1: Linearized vector DNA ( ⁇ ⁇ m : arrow denotes lox site and its orientation) and insert DNA ( ' l ) are mixed in the presence of 15% PEG 6000 to produce conditions of macromolecular crowding, (b) Ligase is added. This would normally result in the generation of long linear concatemers of vector and insert DNA, but if (as depicted here) the vector is dephosphorylated and in large excess, the majority of products will be the vector: :insert: :vector trimolecular hybrids shown. The vector components will be in direct repeat (as shown LEFT) or in inverted repeated (as shown RIGHT) with equal probability.
  • Stage 1 Linearized vector DNA ( ⁇ ⁇ m : arrow denotes lox site and its orientation) and insert DNA ( ' l ) are mixed in the presence of 15% PEG 6000 to produce conditions of macromolecular crowding, (b) Ligase is
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention concerne un vecteur plasmidique p91ox5 contenant un lox de séquence de recombinaison spécifique à un site du système de recombinase lox/Cre des bactériophages P1. La méthode selon l'invention se décompose en deux étapes distinctes. Les vecteurs et fragments d'ADN sont d'abord ligaturés de manière intermoléculaire dans des conditions d'encombrement macromoléculaire (15 % de polyéthylène glycol 6000). Les molécules de recombinaison circulaire sont ensuite excisées des produits de ligature au moyen d'uen recombinase Cre agissant sur des paires de sites lox dans les molécules vectrices directement répétées bordant l'ADN insert. Les produits de recombinaison sont introduits dans les cellules de manière classique par transformation ou électroporation. Les applications de cette technique à la création de bibliothèques d'ADNc et à la récupération de l'ADN de matériaux d'archive sont également présentées.
PCT/GB1994/000272 1993-02-12 1994-02-11 METHODE DE CLONAGE D'ADN A L'AIDE DU VECTEUR Cre-Lox DANS DES CONDITIONS D'ENCOMBREMENT MACROMOLECULAIRE WO1994018333A1 (fr)

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GB9302798.5 1993-02-12
GB939302798A GB9302798D0 (en) 1993-02-12 1993-02-12 Improvements in or relating to cloning dna

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19623203A1 (de) * 1995-11-24 1997-05-28 Max Planck Gesellschaft Virusvektor für den Transfer stabiler Episome
WO2000036088A1 (fr) 1998-12-17 2000-06-22 Yuri Romantchikov Procedes ameliores d'insertion d'acides nucleiques dans des vecteurs circulaires
EP1025217A1 (fr) * 1997-10-24 2000-08-09 Life Technologies, Inc. Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
CN101125873A (zh) * 1997-10-24 2008-02-20 茵维特罗根公司 利用具重组位点的核酸进行重组克隆
US8568982B2 (en) 2006-08-09 2013-10-29 National University Of Singapore Methods of nucleic acid synthesis using particular crowding agents and concentrations
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US9809798B2 (en) 2011-03-11 2017-11-07 National University Of Singapore Pericyte progenitors from peripheral blood

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220009A2 (fr) * 1985-10-07 1987-04-29 E.I. Du Pont De Nemours And Company Recombinaison à site spécifique de l'ADN dans la levure

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220009A2 (fr) * 1985-10-07 1987-04-29 E.I. Du Pont De Nemours And Company Recombinaison à site spécifique de l'ADN dans la levure

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. CHRISTOPHER BOYD: "Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids", NUCLEIC ACIDS RESEARCH., vol. 21, no. 4, 25 February 1993 (1993-02-25), ARLINGTON, VIRGINIA US, pages 817 - 821 *
BRIAN SAUER ET AL.: "The cyclization of linear DNA in Escherichia coli by site-specific recombination", GENE., vol. 70, no. 2, 1988, AMSTERDAM NL, pages 331 - 341 *
PETER UPCROFT ET AL.: "Rapid and efficient method for cloning of blunt-ended DNA fragments", GENE., vol. 51, no. 1, 1987, AMSTERDAM NL, pages 69 - 75 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
DE19623203A1 (de) * 1995-11-24 1997-05-28 Max Planck Gesellschaft Virusvektor für den Transfer stabiler Episome
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
CN101125873A (zh) * 1997-10-24 2008-02-20 茵维特罗根公司 利用具重组位点的核酸进行重组克隆
EP1025217A1 (fr) * 1997-10-24 2000-08-09 Life Technologies, Inc. Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison
EP1025217A4 (fr) * 1997-10-24 2002-11-13 Life Technologies Inc Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison
EP1141239A1 (fr) * 1998-12-17 2001-10-10 Yuri Romantchikov Procedes ameliores d'insertion d'acides nucleiques dans des vecteurs circulaires
EP1141239A4 (fr) * 1998-12-17 2005-04-27 Yuri Romantchikov Procedes ameliores d'insertion d'acides nucleiques dans des vecteurs circulaires
WO2000036088A1 (fr) 1998-12-17 2000-06-22 Yuri Romantchikov Procedes ameliores d'insertion d'acides nucleiques dans des vecteurs circulaires
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
US9309520B2 (en) 2000-08-21 2016-04-12 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US8568982B2 (en) 2006-08-09 2013-10-29 National University Of Singapore Methods of nucleic acid synthesis using particular crowding agents and concentrations
US9809798B2 (en) 2011-03-11 2017-11-07 National University Of Singapore Pericyte progenitors from peripheral blood

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