WO2001025451A2 - Cloning vectors - Google Patents
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- WO2001025451A2 WO2001025451A2 PCT/GB2000/003655 GB0003655W WO0125451A2 WO 2001025451 A2 WO2001025451 A2 WO 2001025451A2 GB 0003655 W GB0003655 W GB 0003655W WO 0125451 A2 WO0125451 A2 WO 0125451A2
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- vector
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- selection
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- GDOPTJXRTPNYNR-UHFFFAOYSA-N CC1CCCC1 Chemical compound CC1CCCC1 GDOPTJXRTPNYNR-UHFFFAOYSA-N 0.000 description 1
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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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
Definitions
- the present invention relates generally to methods and materials, particularly vectors, for use in cloning and recovering nucleic acids .
- DNA molecules lies at the heart of modern molecular biology and typically involves the ligation of one or more DNA fragments or other sequences into suitable vectors, such as plasmids or bacteriophage vectors (1) .
- the DNA fragments to be cloned are usually generated either by restriction endonuclease cleavage of genomic DNA, chemical synthesis or via amplification of DNA or cDNA with a thermostable DNA polymerase (1) .
- the products of the subsequent ligation reaction are introduced into a bacterial host through a range of alternative chemical, biological or physical transformation procedures (2) .
- these bacteria are then grown and observed as colonies on agar plates
- the vector almost always contains an antibiotic
- plasmids containing insert DNA are selected by an appropriate procedure, isolated, and transferred for growth on a suitable culture medium, which is usually liquid.
- positive-selection cloning vectors containing cytotoxic genes (4-11) have simplified the selection of recombinant plasmids by the complete elimination of re- ligated plasmids following transformation of the appropriate bacterial host.
- those plasmids encoding the cytotoxic ccdB gene when a wild-type E. c ⁇ li gyrA+ strain is transformed by such plasmids, the product of the ccdB gene abolishes cell growth (6,7) .
- cytotoxic effect can be alleviated by insertional inactivation of the ccdB gene leading to restoration of cell viability: only those cells containing recombinant plasmids give rise to colonies (6) .
- plasmids encoding cytosine (C-5) - specific DNA methyltransferases (C5 Mtases) are incompatible with E. coli strains proficient in Mcr-mediated restriction
- the present inventors have devised methods and materials for cloning nucleic acid fragments which can be employed to avoid the need for the two step (plating, culturing) methods which form the basis for the systems described above.
- the methods and materials of the present invention permit the selection of hosts containing recombinant plasmids without the necessity of an intermediate plating step and single colony isolation.
- the use of the single step, homogenous system (referred to as a 'non-plating' system) permits the rapid isolation of recombinant plasmids and their inserts.
- the inventors have constructed vectors that combine (i) a gene giving positive selection (e.g. an inactivatable cytotoxic gene) and (ii) a gene which provides a host with protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present (e.g. an antibiotic resistance gene which encodes a product which operates an efflux mechanism) .
- a gene giving positive selection e.g. an inactivatable cytotoxic gene
- a gene which provides a host with protective effect against a selection agent e.g. an antibiotic resistance gene which encodes a product which operates an efflux mechanism
- host cells containing a vector as described above can be used in single step cloning procedures because in appropriate media they have a selective advantage over, in particular, untransformed host cells, and that this advantage (by contrast with the vectors of the prior art) does not appreciably diminish over time.
- vectors are known (e.g. the Zero BackgroundTM Cloning Kit) which comprise a lethal gene (ccdB gene) and an antibiotic resistance marker (Zeocin or Kanamycin) . Instructions provided with these kits require that transformed cells are spread out on LB plates which include the antibiotic in question, which will subsequently be degraded. None of these vectors, or those based in
- Ampicillin are suitable for the single step cloning system of the present invention. Particular aspects of the invention will now be discussed in more detail.
- one aspect of the invention provides a method for isolating a transformed host cell which contains a vector incorporating a nucleic acid insert, which method comprises the steps of:
- a vector which comprises (i) a gene permitting positive selection, which gene is a cytotoxic gene arranged such as to be insertionally inactivated by the incorporation of a heterologous nucleic acid insert into the. vector (ii) a gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present;
- the present invention provides methods for isolating a transformed host cell which contains a vector incorporating a nucleic acid insert, which method does not include the step of growing the host cell on an agar medium and selecting the host cell therefrom.
- the method is one in which transformed host cells which contains a vector incorporating a nucleic acid insert are selected from other host cells in a single homogenous selection step.
- stages prior to plating may be performed in the following timescale. Plas id DNA (cleaved with an appropriate restriction enzyme) is added to an excess of the DNA insert and incubated in the presence of a DNA ligase and appropriate buffer etc. for between 1 and 16 hours, at a temperature of between 4 and 20 degrees C. The products of the reaction are then added to an ice cold suspension of competent E. coli cells, held on ice for between 20-60minute ⁇ , "heat shocked” at 42 degrees C for 2 minutes, and cultured at 37 degrees C for between 30-60 minutes .
- Plating is normally performed by spreading the colonies on an agar plate containing an appropriate antibiotic and incubating at 37 degrees C overnight.
- a vector of the present invention e.g. pMTetl
- the plating stage is omitted and micro- or milligram quantities of recombinant plasmid DNA for plasmid characterisation can be produced by broth culture of in several hours giving an appreciable time saving.
- the direct culture method may facilitate direct storage of desired clones after the addition of glycerol, since there is no requirement for the expansion of selected single colonies into a liquid broth prior to storage.
- vector is defined to include, inter alia, any plasmid, cosmid, phage or the like which can transform a prokaryotic or eukaryotic host, generally by existing extrachromosomally within the host .
- the vector used herein will be an autonomous replicating plasmid with an origin of replication recognised by the host.
- Suitable vectors can be constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, and other sequences as appropriate.
- “Positive selection” is a process whereby something (e.g. a host cell) is selected on the basis of the presence of a particular marker trait or attribute.
- a "cytotoxic” gene is one which can impart a lethal phenotype to an appropriate host cell in which it is expressed i.e. proves lethal to a vector-transformed host.
- “Insertional inactivation" of a gene is the process whereby the introduction of a nucleic acid insert into the vector impairs the activity of the gene or its expression product. Although generally this will mean that the insertion occurs within the promoter or coding sequence of the gene, there is no absolute requirement that this be the case provided that the insertion has the desired effect. Thus if placement of the nucleic acid insert disables the action of the cytotoxic gene, then insertional activation is said to have occurred and restoration of viability (hence positive selection) achieved.
- Heterologous nucleic acid in this aspect is heterologous to the vector used in the process i.e. foreign or external to it. Generally the nucleic acid will generally be DNA or modified DNA. Vectors which contain heterologous nucleic acid inserts may be termed "recombinant" herein.
- a typical protocol for steps (b) - (d) above can be achieved by exposing the vector to a source of heterologous nucleic acid such as to ligate or otherwise incorporate a heterologous nucleic acid insert into the vector at an appropriate cloning site; exposing the ligation product (recombinant vector) to host cells under conditions whereby the vector is taken up by the cells such as to generate a heterogenous population of host cells, some of which contain the vector; exposing the population of cells to a liquid propagation medium comprising the selection agent whereby transformed host cells which contain vector incorporating the nucleic acid insert are selectively grown or propagated in the medium.
- One preferred vector of the present invention i.s the pMTetl vector shown in Fig2 , having a sequence shown in SEQ ID No. 1.
- This includes (i) a modified version of the gene encoding the cytosine specific DNA methyltransferase Mspl , and (ii) a modified form of the pBR322 te A(C) tetracycline resistance gene.
- the combination of these two genes facilitates the selection of recombinant plasmids in broth cultures, thereby eliminating the need for bacterial plating.
- cytotoxic genes for use in a positive selection cassette may be nucleases which have a cytotoxic effect.
- Other examples of cytotoxic genes lude the ccdB gene, when used with a wild-type E. coli gyrA+ strain.
- Other examples, such as the M.AquI and M.Hhal, are discussed in patent application WO97/01639 which is incorporated herein by reference.
- WO97/01639 describes inter alia nucleic acid constructs (e.g. pREVENTl) which encode methyltransferase (e.g. M.AquI) into which inserts can be cloned leading to loss of functionality.
- pREVENTl which encode methyltransferase (e.g. M.AquI) into which inserts can be cloned leading to loss of functionality.
- M.AquI methyltransferase
- the gene is a DNA methyltransferase, more preferably a cytosine (C-5) -specific DNA methyltransferase (C5 Mtase) such as a monospecific (13) or multispecific C5 Mtase gene (4) , when used with an Mcr+ host such as E. coli DH5 ⁇ .
- C5 Mtase cytosine -specific DNA methyltransferase
- Mcr+ host such as E. coli DH5 ⁇
- the gene is the CCGG- specific C5 Mtase gene M .
- MspI which, when active, elicits a potent mcrBC response (12) .
- An example of such a sequence is shown in SEQ ID No . 2.
- mcrBC in which an endonuclease excludes sequences methylated at the 5' position of the Cytosine ring adjacent to a 3' cytosine
- mcrA excludedes methylate.d cytosines followed by guanine bases
- Genes which provide a host cell transformed with the vector with a protective effect against a selection agent, but which do not significantly degrade the selection agent in a medium in which the host is present may be selected from those antibiotic resistance genes which encode products which do not break down the antibiotic in question, but (for instance) operate via a membrane associated antibiotic efflux pump mechanism to lower intracellular concentrations while maintaining, or substantially maintaining, intercellular concentrations . Put another way the gene maintains viability of the transformed cells by keeping the concentration of the liquid medium's antibiotic at a minimum in the cells themselves, while keeping the concentration of antibiotic in the medium relatively constant .
- TetA gene is used. Some of these e.g. TetA(A) to TetA(E) are discussed in (14) . Preferably the TetA(C) gene is used.
- Tetracyclin resistance genes have been used in vectors in the past. Examples include EP0544467 (Eli Lilly and Co.) and EP0502637 (ICI pic) . However these were not positive selection vectors, and were not suitable for the non-plating applications of the present invention.
- derivatives or other variants of the sequences specified above may also be used in the present invention, provided that they encode the requisite activity (e.g. inactivatable cytotoxic activity; gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present; origin of replication recognised by the host; regulatory sequences; polylinker etc).
- cytotoxic activity e.g. inactivatable cytotoxic activity; gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present; origin of replication recognised by the host; regulatory sequences; polylinker etc.
- TetA(c) mutants are discussed in (14) .
- such variants will be substantially homologous to the 'wild type' or other sequence specified herein i.e. will share sequence similarity or identity therewith.
- Similarity or identity may be at the nucleotide sequence and/or encoded amino acid sequence level, and will preferably, be at least about 60%, or 70%, or 80%, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99%. Sequence comparisons may be made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63- 98) .
- Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA; Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA; KTUP word length: 2 for proteins / 6 for DNA. Analysis for similarity can also be carried out using hybridisation.
- T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex (Sambrook et al . , supra) .
- nucleic acid may have been synthesised directly e.g. using an automated synthesiser.
- the nucleic acid molecule may be provided isolated and/or purified from its natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids e.g. a selected DNA fragment separated from a mixture of DNA fragments .
- DNA chromatography such as reverse phase HPLC
- predetermine the size of fragments to be cloned can enhance the efficiency of, and selectivity for, cloning large fragments.
- the insert may be blunt ended, for instance as obtained from a restriction digest or PCR using a proof reading polymerase such as Pfu (Stratagene) or Vent (New England Biolabs) .
- the vector may be adapted so as to facilitate the ligation therein of blunt ended fragments (e.g. may be based on an Invitrogen-TA vector) .
- a selected heterologous nucleic acid DNA fragment, or group of fragments for use in the methods of the present invention may be provided as follows: (a) providing a mixture of DNA fragments; (b) separating the mixture of DNA fragments using RPHPLC (preferably MIPC) on the basis of fragment length.
- RPHPLC preferably MIPC
- a rapid method for cloning and/or amplifying a target heterologous nucleic acid e.g. DNA fragment
- a target heterologous nucleic acid e.g. DNA fragment
- method includes the step of ligating the target fragment into a vector as described above, transforming cells with the ligation product, and growing the cells in a suitable medium.
- Such a method may therefore comprise the steps of: (a) providing a vector which comprises (i) a gene permitting positive selection, which gene is a cytotoxic gene arranged such as to be insertionally inactivated by the incorporation of a heterologous nucleic acid insert into the vector (ii) a gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present;
- a vector suitable for use in the methods disclosed herein, which vector comprises (i) a gene permitting positive selection, which gene is a cytotoxic gene arranged such as to be insertionally inactivated by the incorporation of a heterologous nucleic acid insert into the vector (ii) a gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present.
- the vector may comprise the further components described above.
- the vector comprises a heterologous nucleic acid (a gene of interest, which it is desired to select, clone and/or amplify) inserted therein as described above.
- a host cell comprising a vector of the invention as described herein.
- composition comprising (a) a host cell comprising the vector, and (b) a liquid propagation medium comprising a selection agent as described herein.
- kits comprising a vector of the invention as described herein and at least one further component, which may optionally be selected from (a) a host cell for use with the vector; (b) a liquid propagation medium; (c) a selection agent for use with the medium; (d) one or more primers capable of amplifying or isolating heterologous nucleic acid which is desired to select, clone and/or amplify; (e) one or more test nucleic acid inserts (which may consist of blunt ended double stranded DNA) .
- a system for performing the methods of the invention disclosed herein which system comprises (a) a vector as described herein and at least one further component, which may optionally be selected from (b) a host cell for use with the vector; (c) a liquid propagation medium; (d) a selection agent for use with the medium.
- the system of the present invention which does not require a 'colony picking' step, may be readily automated using standard liquid handling apparatus.
- the system could be an adjunct to a WAVE R TM DNA Fragment Analysis System wherein fractionated fragments are collected, ligated into vectors as described above, and these are used in the methods of the present invention.
- a gene which provides a host cell transformed with a vector comprising that gene with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present, in any one or more of (a) a method disclosed herein; (b) a process for the preparation of a vector, host cell, kit or system disclosed herein.
- a process for preparing a vector comprises the step of ligating (i) a gene permitting positive selection, which gene is a cytotoxic gene arranged such as to be insertionally inactivated by the incorporation of a heterologous nucleic acid insert into the vector (ii) a gene which provides a host cell transformed with the vector with a protective effect against a selection agent, but which does not significantly degrade the selection agent in a medium in which the host is present; or in each case a precursor thereof.
- FIG. 2 Restriction map of pMTetl . All unique restriction sites are indicated. The EcoRV site within the M.MspI gene is used for the insertion of blunt ended DNA molecules.
- SEQ ID No. 1 Complete nucleotide sequence of pMTetl (5086 bp) .
- the Tet gene is located on the vector at position 92- 1279 (396 amino acids) .
- SEQ ID No. 2 / SEQ ID No . 3 Nucleotide sequence and encoded polypeptide (418 amino acids) of modified M.MspI gene. This corresponds to positions 3829-5083 on the vector.
- SEQ ID Nos. 4-17 Various primers of the invention.
- E. coli DH5 and DH5 MCR (for the propagation of modification proficient plasmids) were obtained from Gibco Life Technologies (Paisley, Scotland) . Transformation experiments were carried out as described elsewhere (2) and plasmids encoding M.MspJ were analysed for authentic DNA methylation by overnight digestion with Mspl as described in Landry et al (19) . For transformation reactions in liquid cultures, 4 ⁇ l of a 20 ⁇ l ligation reaction were added to 200 ⁇ l competent cells as described earlier.
- Plasmids were recovered by the alkaline lysis procedure (1) and were analysed using the appropriate combination of restriction enzymes and 1% agarose gels (1) .
- Example 1 construction of pMTetl.
- the first step in the cloning procedure was to remove the unique EcoKV site from within the coding sequence of the tetA (C) gene in pBR322 to produce pBR322 ⁇ RV.
- the M.MspJ coding sequence was excised from pNRT2 , a derivative of pGEX2T harbouring the M.MspI open reading frame between the unique BamHI and EcoRI sites (18) and was inserted into the same sites within the multiple cloning site of pUC19 to generate pUCMspI .
- the sequence which includes a set of unique restriction sites in between the HindiII and
- the tetA (C) gene was inserted into pUCMspI as follows (Fig.lC step 3) .
- pBR322 ⁇ RV was digested with EcoRI and PvuII and the fragment containing the tetA (C) gene was ligated into pUCMspI that had been digested with EcoRI and Seal (both PvuII and Seal leave blunt ends) to produce the recombinant plasmid pMspITetA.
- the plasmid pMspINTetA was produced by ligating the ApaLJ-EcoRI fragment encoding M.MspI from pUCMspIN with the tetA (C) encoding ApaLI-EcoRI fragment from pMspITetA (Fig. ID step 4) .
- the resultant plasmid, pMspINTetA now contains unique ApaLI, EcoRV, EcoRI , HindiII and Sail sites.
- the HindiII site which is upstream of the tetA (C) coding sequence and downstream of the M.MspI gene was removed by cutting the plasmid with HindiII followed by an end-filling reaction catalysed by the Klenow fragment of DNA Polymerase I in the presence of dNTPs and the linearised plasmid was subsequently religated.
- the Sail site (the altered sequence is underlined below) was removed without altering the corresponding TetA(C) primary structure, by site directed mutagenesis using the Quick Change method
- the final plasmid which confers tetracycline resistance to the host and expresses an active Mspl DNA methyltransferase is pMTetl and is shown in Fig.2.
- BRCAl-1 +BRCA1-2 370bp
- BRCAl-1 +BRCA1-4 680bp
- BRCAl-3 +BRCA1-2 890bp
- BRCA2-1 + BRCA2-2 460bp.
- PCRs were performed with Taq polymerase (Promega) using the following programme: 94°C for 90s, 52°C for 2min, 72°C for 3min (30 cycles) .
- Each 50 ⁇ l reaction comprised 200ng genomic DNA, both primers at a final concentration of 0.2 ⁇ M, 1 unit Taq polymerase and 2.5mM MgCl 2 .
- the purified PCR product and the cloning vector were ligated in a 5:1 molar ratio using 4units of T4 DNA ligase: in all cases the cloning vector was linearised with EcoRV. Finally 4 ⁇ l out of the 20 ⁇ l ligation reaction were used to transform 200 ⁇ l of competent E. coli DH5 (2).
- Non-recombinant pUCMspI as a template yields a product of 870bp. All PCR products were analysed on 1% agarose gels (1) .
- the recombinant nature of the plasmids was determined by amplification of an internal stretch of the M.MspJ gene obtained from the general cloning vector pUC19.
- M.MspI gene encoding the DNA sequence recognition domain of the enzyme. Therefore disruption of this section of the coding sequence, as expected, abolishes DNA methyltransferase activity of M.MspI.
- the consequence of this loss of phenotype is that the host encoded McrBC enzyme no longer targets the recombinant plasmid for nucleolytic attack. No colonies were recovered in parallel experiments in which pUCMspI was used to transform E. coli DH5 ⁇ .
- the M.MspI gene was one suitable candidate for the development of a positive selection vector and could be combined with tetracycline resistance in order to develop a cloning system that would eliminate the need for single colony isolation.
- the sequence of steps outlined in Figure 1 show how such a plasmid was assembled.
- the final plasmid is derived from the popular cloning vector pUC19 and combines the gene encoding the M.MspI gene with a modified form of the tetA (C) gene from pBR322.
- the final plasmid construct was modified in order to eliminate restriction sites flanking the M.MspI gene and within the tetA (C) gene.
- the unique EcoRV site in the M.MspI gene was chosen as a general site for the insertion of blunt end DNA molecules generated either by proof-reading DNA polymerases or via restriction digestion with enzymes such as Haelll. Insertion of a range of blunt-end DNA fragments into the EcoRV site of pMTetl abolished the activity of the M.MspI gene, thereby enabling the recombinant (but not intact pMTetl) to escape McrBC-mediated restriction in E. coli DH5 . Expression of tetracycline resistance in liquid culture ensures that the intracellular level of the antibiotic, to which the host is exposed, is kept to a minimum and that the antibiotic concentration in the medium remains essentially constant. This is a consequence of the expression of the membrane- associated antibiotic efflux pump encoded by the tetA (C) gene
- the samples run were pMTetl; a complex mixture of plasmids recovered following ligation of a mixture of ⁇ X174 DNA digested with Haelll and pMTetl; the product of the ligation of a purified 118bp Haelll fragment into pMTetl; a pMTetl recombinant containing a 234bp Haelll fragment and a 603bp Haelll fragment was ligated with pMTetl (plus molecular weight standards) .
- DNASEP R TM column 2 ⁇ g of the DNA fragments dissolved in water was injected on to a preparative scale (50mm x 7.8 mm ID) DNASEP R TM column of bed volume 1.5ml (Transgenomic) and the fragments were eluted at a flow rate of 0.9ml/min.
- a linear gradient of acetonitrile in triethylammonium acetate (TEAA) was applied as follows: buffer A comprised 0. IM TEAA, pH 7.0 (100ml of IM TEAA with 0.25ml acetonitrile, in a total volume of 1 litre) and buffer B comprised 0.
- a positive selection vector based on a combination of a C5 Mtase gene and the tetA (C) gene derived from pBR322 permits the isolation of recombinant plasmids in liquid culture which for the first time eliminates the need to isolate single, antibiotic-resistant colonies and therefore significantly accelerates recombinant plasmid isolation.
- the plasmid particularly facilitates the rapid cloning of DNA molecules generated by proofreading DNA polymerases and restriction digests using enzymes that produce non- cohesive termini.
- this novel cloning vector can be readily employed in conjunction with chromatographic DNA fractionation (15-17) , for the construction of size-selected recombinant molecules.
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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CA002389282A CA2389282A1 (en) | 1999-10-01 | 2000-09-22 | Cloning vectors |
AU75343/00A AU7534300A (en) | 1999-10-01 | 2000-09-22 | Cloning vectors |
EP00964402A EP1218522A2 (en) | 1999-10-01 | 2000-09-22 | Cloning vectors |
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US15707299P | 1999-10-01 | 1999-10-01 | |
US60/157,072 | 1999-10-01 | ||
GB0001716A GB0001716D0 (en) | 2000-01-25 | 2000-01-25 | Cloning vectors |
GB0001716.0 | 2000-01-25 | ||
US65650300A | 2000-09-07 | 2000-09-07 | |
US09/656,503 | 2000-09-07 |
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WO2001025451A2 true WO2001025451A2 (en) | 2001-04-12 |
WO2001025451A3 WO2001025451A3 (en) | 2001-10-18 |
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AU (1) | AU7534300A (en) |
CA (1) | CA2389282A1 (en) |
WO (1) | WO2001025451A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004106526A1 (en) * | 2003-05-22 | 2004-12-09 | Institut Pasteur | Vector for cloning an insert having blunt ends, enabling recombinant clones to be positively selected, method for cloning in said vector, and use of said vector for expressing an insert |
CN107760703A (en) * | 2016-08-23 | 2018-03-06 | 南京理工大学 | A kind of flat end cloning vector pUB857 of zero background and its construction method and application |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1997001639A2 (en) * | 1995-06-27 | 1997-01-16 | The University Of Sheffield | Selection of recombinant molecules |
WO1998038205A1 (en) * | 1997-02-26 | 1998-09-03 | Life Technologies, Inc. | Compositions and methods for positive selection cloning |
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2000
- 2000-09-22 AU AU75343/00A patent/AU7534300A/en not_active Abandoned
- 2000-09-22 CA CA002389282A patent/CA2389282A1/en not_active Abandoned
- 2000-09-22 EP EP00964402A patent/EP1218522A2/en not_active Withdrawn
- 2000-09-22 WO PCT/GB2000/003655 patent/WO2001025451A2/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997001639A2 (en) * | 1995-06-27 | 1997-01-16 | The University Of Sheffield | Selection of recombinant molecules |
WO1998038205A1 (en) * | 1997-02-26 | 1998-09-03 | Life Technologies, Inc. | Compositions and methods for positive selection cloning |
Non-Patent Citations (12)
Title |
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BERNARD P.: "New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers" GENE, vol. 162, no. 1, 30 August 1995 (1995-08-30), pages 159-160, XP004042069 ISSN: 0378-1119 cited in the application * |
CASERTA M. ET AL.: "Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 262, no. 10, 5 April 1987 (1987-04-05), pages 4770-4777, XP002163571 * |
FIVES-TAYLOR P.M. ET AL.: "Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae" INFECTION AND IMMUNITY, vol. 55, no. 1, January 1987 (1987-01), pages 123-128, XP002163241 * |
LEWIS M.K. AND THOMPSON D.V.: "Efficient site directed in vitro mutagenesis using ampicillin selection" NUCLEIC ACIDS RESEARCH, vol. 18, no. 12, 1990, pages 3439-3443, XP002049079 ISSN: 0305-1048 * |
MATIN M.M. AND HORNBY D.P.: "A positive selection vector combining the tetracycline resistance that eliminates the need for bacterial plating" ANALYTICAL BIOCHEMISTRY, vol. 278, 1 February 2000 (2000-02-01), pages 46-51, XP002163244 * |
MAYER M.P.: "A new set of useful cloning and expression vectors derived from pBlueScript" GENE, vol. 163, no. 1, 22 September 1995 (1995-09-22), pages 41-46, XP004041950 ISSN: 0378-1119 * |
NWANKWO D.O. AND WILSON G.G.: "Cloning and expression of the MspI restriction and modification genes" GENE, vol. 64, 1988, pages 1-8, XP002163240 * |
O'CONNOR C.D. AND HUMPHREYS G.O.: "Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors" GENE, vol. 20, 1982, pages 219-229, XP000990434 * |
PUCCI M.J. AND MACRINA F.L.: "Cloned gtfA gene of Streptococcus mutans LM7 alters glucan synthesis in Streptococcus sanguis" INFECTION AND IMMUNITY, vol. 48, no. 3, June 1985 (1985-06), pages 704-712, XP002163242 * |
RALEIGH E.A. AND WILSON G.: "Escherichia coli K-12 restricts DNA containing 5-methylcytosine" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 83, 1 December 1986 (1986-12-01), pages 9070-9074, XP000653066 ISSN: 0027-8424 * |
WINTER R.B. AND GOLD L.: "Overproduction of bacteriophage Qbeta maturation (A2) protein leads to cell lysis" CELL, vol. 33, no. 3, 3 July 1983 (1983-07-03), pages 877-885, XP000990376 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004106526A1 (en) * | 2003-05-22 | 2004-12-09 | Institut Pasteur | Vector for cloning an insert having blunt ends, enabling recombinant clones to be positively selected, method for cloning in said vector, and use of said vector for expressing an insert |
CN107760703A (en) * | 2016-08-23 | 2018-03-06 | 南京理工大学 | A kind of flat end cloning vector pUB857 of zero background and its construction method and application |
Also Published As
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WO2001025451A3 (en) | 2001-10-18 |
AU7534300A (en) | 2001-05-10 |
EP1218522A2 (en) | 2002-07-03 |
CA2389282A1 (en) | 2001-04-12 |
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