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WO1992007949A1 - Procede de production in vitro de proteine a partir d'une sequen ce d'adn sans clonage - Google Patents

Procede de production in vitro de proteine a partir d'une sequen ce d'adn sans clonage Download PDF

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Publication number
WO1992007949A1
WO1992007949A1 PCT/US1991/008291 US9108291W WO9207949A1 WO 1992007949 A1 WO1992007949 A1 WO 1992007949A1 US 9108291 W US9108291 W US 9108291W WO 9207949 A1 WO9207949 A1 WO 9207949A1
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Prior art keywords
promoter
sequence
universal promoter
universal
dna
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PCT/US1991/008291
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English (en)
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David Ervin Lanar
Kevin C. Kain
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The United States Of America, As Represented By The Secretary Of The Army
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Publication of WO1992007949A1 publication Critical patent/WO1992007949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
    • 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

Definitions

  • This invention pertains generally to the field of molecular biology and particularl to the production of RNA and protein in vitro.
  • the cloning procedure requires isolation of the DNA fragment, genetic engineering of proper ends onto the molecule, ligation into a enzyme digested prepared plasmid, transformation of that ligated DNA into bacteria, selection of transformants and then purification of the ligated plasmid.
  • This plasmid must then be digested with a suitable enzyme at a point in or after the gene in order to end transcription. Plasmid is then purified and in vitro transcribed into mRNA. This mRNA is translated into protein.
  • the disadvantage of the above described in vitro manipulations is that the entire procedure can take as long as two or more months for each single construct that is made.
  • One approach, for example, to express a gene fragment would involve the following steps. First the DNA fragment would have to be purified.
  • a ribosome binding site has to be added to the fragment and if desired, a sequence which can act as an enhancer of translational activity can be added.
  • the modified DNA fragment is ligated into the prepared vector plasmid by the use of the enzyme T-4 ligase and transformed into bacteria by electroporation or absorption. Bacteria which have taken up plasmids are identified by plating on nutrient agar plates containing an antibiotic to which resistance is confirmed by an antibiotic resistance gene on the same plasmid. Single colonies are isolated and analyzed for plasmids with inserts of the desired DNA fragment.
  • RNA polymerase such as T7 RNA polymerase.
  • T7 RNA polymerase a RNA polymerase
  • the other reagents needed are the linearized DNA template, salts, buffer and ribonucleotides. Commercial kits are available to perform this synthesis.
  • RNA construct that contains a site of RNA polymerase binding, an enhancer of translational activity, a ribosome binding site and a "start codon" or ATG sequence in front of any desired DNA fragment in order to obtain RNA copies which can be translated in vitro. It is, therefore, also, an object of the present invention to make these gene fragments quickly and easily, by one reasonable skilled in the art of recombinant DNA technology, without the need to clone the DNA fragment into a plasmid vector.
  • FIG. 1 is the double stranded sequence of the first construct of universal promoter (UP-1) showing the site of binding of the T7 RNA polymerase, the untranslated leader sequence (UTL) from alfalfa mosaic virus (AMV) and the triplet codons for the amino acids MET and ALA.
  • FIG. 2 is the double strand sequence of the what is generated when UP-1 is spliced to DNA fragment of interest by SOE using primer H3T7 as the forward primer.
  • FIG. 3 is the double strand sequence of the second construct of a universal promoter (UP-3) which differs from UP-1 by the modification of the 3' end.
  • FIG. 1 is the double stranded sequence of the first construct of universal promoter (UP-1) showing the site of binding of the T7 RNA polymerase, the untranslated leader sequence (UTL) from alfalfa mosaic virus (AMV) and the triplet codons for the amino acids MET and ALA.
  • FIG. 2 is the double strand sequence of
  • FIG. 4 is the double strand sequence of the what is generated when UP-3 is spliced to DNA fragment of interest by SOE using primer H3T7 as the forward primer.
  • FIG. 5 is the sequences of the oligonucleotide primers used in the construction and testing of the universal promoters
  • FIG. 6 is a diagrammatic outline of the steps used in the expression PCR reactions. The double stranded DNA and oligomers are represented by lines and arrows indicating the 5' to 3' orientation. Oligomers are denoted by bold upper-case letters and amplified products by the steps 1-3.
  • the univeral promoter is first made from primers pUP1 and pUP3 (step 1).
  • the gene to be expressed is amplified from genomic or plasmid DNA using primers A and B. Because primer A has nucleotides at its 5' end complimentary to the 3 * end of the universal promoter the amplified product (step 3) has a region homologous to the 3' end of the universal promoter at its 5' end.
  • the products of step 1 and step 2 are mixed with primer H3T7 primer B (boxed reaction) and amplified in a two step PCR analogous to splicing by overlap extension. Initially no primers are added and each DNA strand acts as primer and a template for the other to produce the recombinant molecule composed of the T7 promoter and the untranslated leader sequence spliced in frame to the gene of interest.
  • FIG. 7 is an immunoprecipitation of E-PCR product of a "repeatless" gene construct of the P. falciparium CS gene.
  • mRNA was produced by the E-PCR method and translated in a rabbit reticulocyte lysate cell-free system in the presence of [ 35 S] and analyzed by 0.1% SDS - 15% PAGE and autoradiography (lane a).
  • Translated repeatless CS protein was immunoprecipitated only by antisera against the repeatless portion of the protein (lane c) and not by antibodies to the repeat region (lane b) or by antibodies specific for the CS protein of P.
  • Fig. 8. is the analysis of two different E-PCR constructs of the EBA-175 gene. Immunoprecipitation and specific binding to RBC's are shown.
  • the EBA-175 mRNA's produced by the E-PCR method were translated in a wheat germ cell-free system in the presence of [ 3 H]leucine and analyzed by 0.1% SDS - 15% PAGE and autoradiography (lanes 1 and 3).
  • Translated protein was immunoprecipitated by antipeptide 4 sera (lane 2) which recognized a peptide within this construct. Full length protein product made in lane 3 bound to RBC's susceptible to invasion by P. falciparum merozoites (lane 4).
  • the above objects and advantages of the present invention are achieved by the use of the "universal promoter" in the procedure described by us as expression polymerase chain reaction (E-PCR).
  • E-PCR expression polymerase chain reaction
  • This invention allows selective in vitro transcription of DNA in accordance with the invention without the need to clone the piece of DNA into a plasmid vector.
  • the active polymerase binding site (FIG 1 , positions -1 to -17) described in this invention is the one for viral T7 RNA polymerase, however, any promoter site may be used that corresponds to the RNA polymerase that will be employed for the transcription of the DNA.
  • promoter sites for suitable polymerases are those for the SP6 polymerase, the T3 or N4 phage polymerase or the ghl promoter. Many other polymerases and the promoters they recognize can be used in accordance with the invention.
  • the untranslated leader (UTL) sequence between the T7 promoter and the initi ATG codon (FIG. 1 , positions + 1 to +38) is derived from the coat protein mRNA of th alfalfa mosaic virus (AMV). In vitro translation of mRNA is often dependent on the presence of, and characteristics of, an UTL sequence 5 * to the initiation codon.
  • Footprint analysis indicates that the sequence of the upstream fragments is not critical except that at least 5 nucleotides are probably needed to stabilize the protein-DNA interaction of the polymerase with the promoter site.
  • the original construction of the UP-1 (FIG.1) contained only 3 nucleotides upstream from the -17 nucleotide. This construct did not give efficient transcription of the downstream DNA into RNA.
  • primer H3T7 (FIG. 5) which adds nine nucleotides upstream of the -17 position. This primer was used in the third step of the E-PCR reaction (E ⁇ XAMPLE 3, below) and resulted in UP-2 which gave increased transcriptional activity.
  • nucleotides selected for this extension contain the site of the Hind III restriction endonuclease only for future considerations of cloning the UP, and are not meant to be specifically needed. Further minor modifications in the universal promoter sequence were made at the 3 * end to facilitate primer design for the SOE reaction (see EXAMPLE 1).
  • UP universal promoter
  • UTL untranslated leader
  • EXAMPLE 1 Construction of the Universal promoter A universal promoter-1 having the sequence shown in FIG. 1 was constructed by primer-dimer formation in a polymerase chain reaction (Saika, R.K., et al. (1988) Science 239:487-491; Browning, K.S., (1989) Amplifications 3:14-15.) from the primers pUP1 and pUP2 shown in FIG. 5.
  • the two primers pUP1 and pUP2 were synthesized on an Applied Biosystems 380B DNA synthesizer, deprotected by ammonium hydroxide treatment and desalted by passage over a Pharmacia PD-10 column containing Sephadex G-25 as described (Jayaraman, K., (1987) Biotechiques 5(7):627). They were added together in a PCR reaction and because their last five 3' nucleotides were complementary, amplified each other in a primer-dimer formation reaction. The double stranded universal promoter-1 was applied to an agarose gel to electrophorese away unextended single stranded primers.
  • the double stranded universal promoter- 1 was visualized by ethidium bromide staining and the area of the gel containing the universal promoter-1 band was excised and stored in a 1.5 ml polypropylene microcentrifuge tube.
  • the sequence of pUP1 was designed to include the T7 RNA polymerase binding site and part the of UTL sequence of AMV.
  • the sequence of pUP2 was designed to include the remaining complementary downstream sequence of the AMV UTL with a five base overlap complementary to the UTL sequence of pUP1, and a start translation 'ATG' codon.
  • An additional feature was the design of a Nco 1 restriction endonuclease site around the 'ATG' codon to facilitate future cloning if desired (FIG 1.).
  • the universal ⁇ romoter-3 was redesigned to include nine base pairs upstream from the -17 site of the T7 promoter binding site (Fig 4) and called universal promoter-4.
  • 100 pmols of pUP1 and pUP2 were added in a final 100ul reaction containing 200 uM each dNTP (dATP, dGTP, dCTP, dTTP), 10ul 10X reaction buffer (10X reaction buffer consists of 100mM Tris-HCI, pH8.3, 500 mM KCI, 15 mM MgCI 2 , 0.01% (w/v) gelatin) and 2.5 units Taq DNA polymerase.
  • the reaction was amplified by 20 cycles each of 2 min at 94° C, 2 min at 50° C, 2min at 72° C. The last cycle was followed by an incubation at 72° C for 7 min.
  • the reaction was extracted one time with 100 ul of chloroform, precipitated by adding 50 ul of 7.5M ammonium acetate and 2 vols of 100% ethanol and placing at -20° C for 30 min.
  • the sample was centrifuged, the precipitate washed with cold 80% ethanol and resuspended in 20 ul of TE (10mM Tris, pH 8.0, 1mM EDTA), and loaded onto an agarose gel consisting of 2% NuSieve agarose, 1% Seakem agarose, 0.5ug/ml ethidium bromide, 1X TAE buffer (10X Buffer is 0.4M Tris Base, 0.2M sodium acetate, 10mM EDTA, pH7.2).
  • T e gel was electrophoresed for 30min in 1X TAE buffer and the band excised and stored in a 1.5ml polypropylene microcentrifuge tube at -20° C.
  • EXAMPLE 2 PCR of the DNA of interest Synthesis of the gene segment of interest was done by the standard PCR technique. Oligonucleotide primers for the PCR reaction were synthesized and purified as described in EXAMPLE 1. The key to designing the primers required for this invention was to include on the 5' end of the splicing primer A (FIG. 5) the same sequence of 7 nucleotides as the last 7 nucleotides on the 3' end of the universal promoter-1 with the addition of two bases so the gene segment of interest was in the correct reading frame with the start of translation signal (ATG) of the universal promoter-1. The bases chosen were selected to give the amino acid LEU upon translation.
  • the 5' end of the splicing primer A needed to be the same as the last 9 nucleotides on the 3' end of the universal promoter.
  • the remaining 18 nucleotides of splicing primer A were specific to the DNA segment of interest, in this example a gene segment on the codin strand of EBA-175 (Sim, et al 1990).
  • the sequence of the reverse primer was specific to a downstream segment of the same gene but on the complementary strand.
  • SOE-PCR overlap extension PCR
  • the splicing primers A and the reverse primer B were synthesized on an Applied Biosystems DNA synthesizer (model 380B) and deblocked with ammonium hydroxide treatment and desalted over a PD10 column.
  • PCR reaction 50 pmol of each primer were added in a final 100ul reaction with 10 ng template DNA (the EBA-175 gene cloned into a plasmid), 200 uM each dNTP, 10ul 10X reaction Buffer and 2.5 units Taq DNA polymerase.
  • the reaction was amplified in an automated thermal cycler (Perkin Elmer Cetus) using 25 cycles (each consisting of 2 min at 94° C, 2 min at 50° C, 2 min at 72° C) followed by a 7 minute incubation at 72° C.
  • the PCR products were separated on a 2% LMP NuSieve agarose gel and the DNA bands were excised and stored at 4° C until spliced to the universal promoter by a SOE-PCR reaction.
  • EXAMPLE 3 Splicing bv overlap extension of the UP to the ⁇ ene fragment of interest
  • the agarose containing the universal promoter made in EXAMPLE 1 and agarose containing the gene of interest, made in EXAMPLE 2 were melted at 60° C, and 2 ul of melted agarose containing 25ng of each DNA were added together, without primers, in a single PCR reaction of a final 90 ul volume containing 9 ul 10X reaction buffer and 200 uM each dNTP, 1.3 units Taq polymerase for 15 cycles (each consisting of 2 min at 94° C, 2 min at 25° C, 2 min at 72° C).
  • primers H3T7 and the reverse primer B (FIG. 5) and an additional 1.3 units of Taq polymerase were added in 10 ul 1X reaction buffer and the amplification continued for 25 cycles (each consisting of 2 min at 94° C, 2 min at 55° C, 2 min at 72° C).
  • the PCR products were extracted with chloroform, precipitated with ethanol, and resuspended in 10 ul RNAse-free water.
  • EXAMPLE 4 In vitro transcription of DNA templates The DNA of interest spliced to the universal promoter was transcribed into mRNA in vitro with the use of a commercially available in vitro transcription kit (Pro- Mega, Madison, Wl). This reaction makes mRNA molecules that have at their 5' ends a site for ribosome binding, an enhancer of translational activity and a start AUG codon in correct reading frame with the desired sequence downstream from it. Regions transcribed are not dependent on the presence of restriction enzyme sites, but are dictated by the selection of the original primers in EXAMPLE 2. This allows all molecules to initiate and end at any desired point within the open reading frame of the gene.
  • Extra primers left from the PCR reaction do not have to be removed as they do not interfere with the transcription reaction and do not bind to the native T7 RNA polymerase.
  • One microliter of DNA template produced by E-PCR (EXAMPLE 3) was added to a 50 ul transcription reaction containing 40mM Tris HCL pH 8.0; 8mM MgCI 2 ; 2mM spermidine; 10 mM NaCI; 10 mM DTT; 40 units of RNasin (Pro-Mega); 500 uM each of ATP, CTP, GTP, and UTP; and 25 units of T7 RNA polymerase (Pro-Mega).
  • the reaction was incubated at 37°C for 60 minutes.
  • the DNA template was digested with unit of RQ1 DNase (Pro-Mega) at 37°C for 15 minutes, followed by phenol extraction, ethanol precipitation, and resuspension in 10 ul RNase-free water.
  • EXAMPLE 5 In vitro translation of protein
  • the mRNA transcribed from the E-PCR DNA template can be translated in vitro using a variety of commercially available systems that include rabbit reticulocyte lysate wheat germ extract or bacterial extract. These systems each contain the endogenous cellular components necessary for protein synthesis: ribosomes; tRNA; and initiation, elongation, and termination factors/ A mixture of amino acids is added, one or more o which can be labeled with a radioactive marker to allow quantitation and analysis of th protein product.
  • the optimum potassium acetate and magnesium acetate levels for each particular mRNA should be determined for highly efficient translation of the mRNA.
  • the mRNA may be injected into in vivo translation systems such as frog oocytes. Therefore, following fairly standard protocols radiolabeled protein can be produced which can be used in a functional assay such as precipitation with specific antibody or binding to a specific receptor.
  • a functional assay such as precipitation with specific antibody or binding to a specific receptor.
  • the mRNA was heated to 67° C for 10 min and immediately cooled on ice. This increases the efficiency of translation, especially of GC-rich mRNA, by destroying local regions of secondary structure.
  • the reagents of the translation mixture were added in a 0.5ml polypropylene microcentrifug ⁇ tube (35ul nuclease treated lysate, 7ul water, lul RNasin ribonuclease inhibitor (at 40u/ul), 1ul mM amino acid mixture (minus leucine), 1 ul mRNA substrate, 5ul 3 H-leucine (100-200Ci/mmole) at 5mCi/ml). This reaction is incubated at 30° C for 60 min.
  • EXAMPLE 6 Immunoprecipitation of in vitro translated protein with specific antibody This example illustrates the use of this method to check that a cloned gene is in the correct reading frame or that mutations have not been introduced during cloning manipulations that could alter the reading frame of the expected recombinant product.
  • the protein product of a gene cloned in the correct reading frame produces an epitope that is recognized by antibodies specific to epitopes on that protein.
  • the DNA segment of interest can be amplified directly, as in EXAMPLE 2, from a bacterial colon and that PCR product can be spliced to the universal promoter-3 as in EXAMPLE 3; transcribed into mRNA as in EXAMPLE 4; and translated into protein as in EXAMPLE 5.
  • This protein can then be immunoprecipitated with antibodies against epitopes specific to the protein (FIG 7).
  • a DNA construct was made of the gene for the circumsporozo ' rte protein of Plasmodium falciparum that did not code for the internal repeated peptides.
  • This construct was cloned into a plasmid vector, pADE171 and transformed into the bacteria Salmonella typhimurium. After construction and transformation it was of interest to determine whether or not the gene could encode the correct "repeatless” protein. Production of the protein in Salmonella is minimal under in vitro growth conditions. Therefore, E-PCR was employed to answer the question. A single colony of bacteria on an agar plate was touched with a sterile toothpick and adherent bacteria were lysed by placing the toothpick into 10 ul of 0.1 N NaOH for 10 min at room temperature. The solution was neutralized with 10 ul of 0.5M Tris, pH 7.5 and the 20 ul added to 980 ul of water.
  • T7CS splicing primer
  • RAS 2 reverse primer
  • the DNA was spliced to the universal promoter-3 as in EXAMPLE 3; transcribed into mRNA as in EXAMPLE 4; and translated into protein as in EXAMPLE 5 in the presence of 35 S-methionine. About 100,000 cpm were added to antibody that was specific to either the repeat region (which should not be made in this construct), the non repeat region, the CS gene product of Plasmodium berghei, or normal rabbit antibody. The mixture was allowed to react for 1 hr at room temperature and then Protein A-Sepharose beads were added for 1 hr.
  • lane A As shown in FIG 8, lane A, several size products are translated from the mRNA, most probably due to incomplete synthesis by the ribosomes or truncated mRNA molecules. However, as shown in lane B, only full length polypeptide bound to red blood cells.

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Abstract

On peut obtenir une production in vitro de protéine à partir de séquences d'ADN à l'aide d'une nouvelle technique expérimentale unique appelée expression-amplification enzymatique du génome (E-PCR) ne nécessitant pas le clonage du segment d'ADN étudié en n'importe quel plasmide ou phage vecteur. On a mis au point un promoteur universel contenant une séquence leader non traduite provenant du virus de la mosaïque de la luzerne en aval du promoteur de bactériophage T7. Lorsque le promoteur universel est épissé à un segment d'ADN de manière appropriée, il produit un gabarit adapté à une transcription et une traduction in vitro. L'ADN à exprimer est premièrement amplifié par l'amplification enzymatique du génome (PCR) à l'aide d'une amorce en 5' comprenant une zone homologue à l'extrémité 3' du promoteur universel. Le promoteur universel et ce fragment d'ADN sont mélangés et réamplifiés dans une réaction analogue à l'épissage par extension en chevauchement, produisant un promoteur universel modifié lié à une séquence d'ADN pouvant alors être transcrite et traduite efficacement in vitro sans autre traitement.
PCT/US1991/008291 1990-11-05 1991-11-05 Procede de production in vitro de proteine a partir d'une sequen ce d'adn sans clonage WO1992007949A1 (fr)

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Cited By (16)

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WO1995017523A3 (fr) * 1993-12-15 1995-09-28 Univ Johns Hopkins Diagnostic moleculaire de la polypose adenomateuse hereditaire
FR2786788A1 (fr) * 1998-12-08 2000-06-09 Proteus Procede de criblage de substances capables de modifier l'activite d'une ou plusieurs proteines cibles ou d'un ensemble cible de proteines exprimees in vitro
FR2786787A1 (fr) * 1998-12-08 2000-06-09 Proteus Methode d'analyse in vitro d'un phenotype connu a partir d'un echantillon d'acides nucleiques
FR2786789A1 (fr) * 1998-12-08 2000-06-09 Proteus Methode de detection in vitro d'une sequence d'acide nucleique cible dans un echantillon d'acide nucleique
DE10113265A1 (de) * 2001-03-16 2002-10-02 Rina Netzwerk Fuer Rna Technol Verfahren zur präparativen Herstellung von langen Nukleinsäuren mittels PCR
WO2002064773A3 (fr) * 2001-02-09 2003-05-08 Hans-Joachim Mueller Procede de selection a haut rendement de composes candidats par synthese directe a haut rendement de polypeptides recombinants
US6818396B1 (en) 2000-11-28 2004-11-16 Proteus S.A. Process for determination of the activity of a substance using an in vitro functional test
US6911307B1 (en) 1998-12-08 2005-06-28 Proteus S.A. Method of detection in vitro of a target substance in a sample comprising the labelling of said substance with a reporter gene and with the sequences necessary for the expression of said reporter gene in vitro
US7195895B2 (en) * 2001-07-02 2007-03-27 Riken Method of producing template DNA and method of producing protein in cell-free protein synthesis system using the same
US8226976B2 (en) 2007-11-05 2012-07-24 Riken Method of using cell-free protein synthesis to produce a membrane protein
US8445232B2 (en) 2004-11-17 2013-05-21 Riken Cell-free system for synthesis of proteins derived from cultured mammalian cells
US8603774B2 (en) 2002-11-28 2013-12-10 National Food Research Institute Extract of E. coli cells having mutation in ribosomal protein S12, and method for producing protein in cell-free system using the extract
US8664355B2 (en) 2004-11-19 2014-03-04 Riken Cell-free protein synthesis method with the use of linear template DNA and cell extract therefor
WO2016057951A3 (fr) * 2014-10-09 2016-06-02 Life Technologies Corporation Oligonucléotides crispr et édition de gènes
US11618777B2 (en) 2015-07-31 2023-04-04 Shigeyuki Yokoyama Method of manufacturing membrane protein and utilization thereof
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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Cited By (31)

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Publication number Priority date Publication date Assignee Title
WO1995017523A3 (fr) * 1993-12-15 1995-09-28 Univ Johns Hopkins Diagnostic moleculaire de la polypose adenomateuse hereditaire
US5709998A (en) * 1993-12-15 1998-01-20 The Johns Hopkins University Molecular diagnosis of familial adenomatous polyposis
US5760207A (en) * 1993-12-15 1998-06-02 The Johns Hopkins University Primers for amplifying APC gene sequences
WO2000034513A1 (fr) * 1998-12-08 2000-06-15 Proteus (S.A.) METHODE DE DETECTION IN VITRO D'UNE SUBSTANCE CABLE DANS UN ECHANTILLON COMPRENANT LE MARQUAGE DE LADITE SUBSTANCE PAR UN GENE RAPPORTEUR ET PAR LES SEQUENCES NECESSAIRES A L'EXPRESSION DUDIT GENE RAPPORTEUR $i(IN VITRO)
FR2786787A1 (fr) * 1998-12-08 2000-06-09 Proteus Methode d'analyse in vitro d'un phenotype connu a partir d'un echantillon d'acides nucleiques
FR2786789A1 (fr) * 1998-12-08 2000-06-09 Proteus Methode de detection in vitro d'une sequence d'acide nucleique cible dans un echantillon d'acide nucleique
US6911307B1 (en) 1998-12-08 2005-06-28 Proteus S.A. Method of detection in vitro of a target substance in a sample comprising the labelling of said substance with a reporter gene and with the sequences necessary for the expression of said reporter gene in vitro
WO2000034512A1 (fr) * 1998-12-08 2000-06-15 Proteus (S.A.) Methode de detection et/ou de quantification d'une fonction connue a partir d'un echantillon d'acides nucleiques
WO2000034514A1 (fr) * 1998-12-08 2000-06-15 Proteus (S.A.) PROCEDE DE DETERMINATION DE L'ACTIVITE D'UNE SUBSTANCE METTANT EN OEUVRE UN TEST FONCTIONNEL $i(IN VITRO)
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