WO2000053803A1 - FRAGMENT NUCLEOTIDIQUE, SONDE, AMORCE, REACTIF ET PROCEDE DE DETECTION D'UNE SEQUENCE NUCLEOTIDIQUE DERIVEE DE L'ORIGINE DE REPLICATION DE pBR322 - Google Patents
FRAGMENT NUCLEOTIDIQUE, SONDE, AMORCE, REACTIF ET PROCEDE DE DETECTION D'UNE SEQUENCE NUCLEOTIDIQUE DERIVEE DE L'ORIGINE DE REPLICATION DE pBR322 Download PDFInfo
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- WO2000053803A1 WO2000053803A1 PCT/FR2000/000543 FR0000543W WO0053803A1 WO 2000053803 A1 WO2000053803 A1 WO 2000053803A1 FR 0000543 W FR0000543 W FR 0000543W WO 0053803 A1 WO0053803 A1 WO 0053803A1
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the present invention relates to the detection, using nucleotide probes, of nucleotide sequences derived from the origin of replication of the vector pBR322, present in gene therapy vectors.
- Gene therapy is defined as the transfer of genetic information of therapeutic interest into a host cell or organism.
- the first protocol applied to humans was initiated in the United States in September 1 990 on a genetically immunodeficient patient due to a mutation affecting the gene coding for Adenine Deaminase (ADA). It was a question of correcting or replacing the defective gene whose dysfunction is at the origin of a genetic disease by a functional gene. The relative success of this first experiment encouraged the development of this technology which has since been extended to the treatment of other diseases, both genetic and acquired (cancers, infectious diseases like AIDS ...) with the aim of delivering in situ therapeutic genes. Most strategies use vectors to convey the therapeutic gene to its cell target.
- the aim of the present invention is to provide a reliable, sensitive detection test applicable to a large number of vectors used in gene therapy protocols.
- Plasmid pBR322 (GenBank Accession Number 27-4902-01), derived from the wild-type plasmid ColE1, is commonly used as starting material for the preparation of many gene therapy vectors.
- PBR322 is well characterized and has more than thirty unique restriction sites, which makes it particularly advantageous in the design of new vectors [F. Bolivar et al, Gene 2, 95 (1,977) and N. Watson, Gene, 70, 399 (1,988)].
- the pBR322 region comprising its origin of replication has served as the basis for the construction of numerous gene therapy vectors.
- a tool and a method for detecting a nucleotide sequence derived from the origin of replication of said vector applicable to any vector derived from pBR322.
- a first object of the invention is a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, under conditions of high stringency, to the sequence defined by SEQ ID NO: 1 or to its complementary sequence.
- said sequence comprises at least twelve contiguous nucleotide motifs of a sequence chosen from SEQ ID NO: 1 and its complement.
- such a fragment preferably constitutes a probe.
- sequence SEQ ID NO: 1 described at the end of the description in the sequence list corresponds to the complementary DNA sequence starting at nucleotide 2140 and ending at nucleotide 3145 of the sequence of the plasmid available in databases (GenBank, www .ncbi.nlm.nih.gov /) under the access number J01 749, and SEQ ID NO: 2 corresponds to that of the plasmid ColE1, for the same region (access number J01 566 in the same bank).
- vector derived from pBR322 is intended to denote the constructs of recombinant nucleic acid of which at least the origin of replication is derived from pBR322.
- a vector will consist of a plasmid, it being moreover understood that said vector may also comprise nucleic sequences heterologous to pBR322 and in particular nucleic sequences or constructs of nucleic acids of therapeutic interest.
- the gene of therapeutic interest codes for a
- Antisense RNA a ribozyme, or even a polypeptide of interest. It can come from a eukaryotic organism, a prokaryote from a parasite or a virus. It can be isolated by any conventional technique in the field of art (by cloning, PCR, chemical synthesis, etc.). It can be of genomic type (comprising all or part of all the introns), of complementary DNA type (cDNA, devoid of intron) or of mixed type (minigene). Furthermore, the polypeptide for which it codes can be (i) intracellular, (ii) incorporated into the membrane of the host cell or (iii) secreted. It may be a polypeptide as found in nature (native), a portion of it (truncated), a mutant having in particular biological properties improved or modified or of a chimeric polypeptide originating from the fusion of sequences of various origins.
- chemokines MIP-1 a, MIP-1 b, RANTES, DC-CK1, MDC, MCP1 (monocyte chemoattraction protein), IP10, etc.
- cytokines interferon a, b or g, interleukin (IL), in particular IL-2, IL-6, IL-10 or even IL-1 2, colony stimulating factor (GM-CSF , C-CSF, M-CSF)
- cytokines interferon a, b or g, interleukin (IL), in particular IL-2, IL-6, IL-10 or even IL-1 2, colony stimulating factor (GM-CSF , C-CSF, M-CSF)
- cellular receptors especially recognized by the HIV virus
- receptor ligands receptor ligands
- coagulation factors Factor VIII, Factor IX, thrombin, protein C
- factors growth factors proangiogenic factors
- FGF for Fibroblast Growth Factor
- VEGF for Vascular Endotheli
- a functional copy of the defective gene will be used, for example a gene coding for factor VIII or IX in the context of hemophilia A or B, dystrophin (or minidystrophin) in the framework of Duchenne and Becker's myopathies, insulin in the context of diabetes, CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator) in the context of cystic fibrosis.
- factor VIII or IX in the context of hemophilia A or B
- dystrophin or minidystrophin
- CFTR protein Cystic Fibrosis Transmembrane Conductance Regulator
- TK-HSV-1 thymidine kinase of virus simplex virus l herpes 1
- ricin ricin
- cholera toxin diphtheria
- an immunoprotective polypeptide for example a CD4-immunoglobulin IgG hybrid; Capon et al., 1 989, Nature 337, 525-531; Byrn et al., 1 990, Nature 344, 667-670
- an immunotoxin for example fusion of the antibody 2F5 or of the immunoadhesin CD4-2F5 to angiogenin; Kurachi et al., 1 985, Biochemistry 24, 5494-5499
- a trans-dominant variant for example fusion of the antibody 2F5 or of the immunoadhesin CD4-2F5 to angiogenin
- said vectors or nucleic acid construct usable in gene therapy can be in their naked form (Wolff et al., 1 990, Science 2, pages 1 465-1 468.), associated with liposomes, cationic lipids, cationic polymers, peptides, polypeptides.
- the literature relating to vectors usable in gene therapy provides a large number of examples of such vectors (see for example Robbins et al, 1 988, Tibtech, 1 6, 34-40 and Rolland, 1 998, Therapeutic Drug Carrier Systems, 1 5, 143-1 98).
- nucleotide sequence means a linear or circular, single-stranded or double-stranded DNA sequence
- a "nucleotide fragment” and an “oligonucleotide” are two synonymous terms designating a chain of motifs nucleotides characterized by the informational sequence of natural (or possibly modified) nucleic acids and capable of hybridizing, like natural nucleic acids, with a complementary or substantially complementary nucleotide fragment, under predetermined conditions; the chain may contain nucleotide units with a structure different from that of natural nucleic acids; a nucleotide fragment (or oligonucleotide) generally contains at least 12 nucleotide motifs and can be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis;
- a nucleotide motif is derived from a monomer which may be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is deoxy-2-ribose, in RNA the sugar is ribose; depending on whether it is DNA or RNA, the nitrogen base is chosen from adenine, guanine, uracil, cytosine, thymine; or the monomer is a nucleotide modified in at least one of the three constituent elements; for example, the modification may take place, either at the base level, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6 -purine, bromo-5-deoxyuridine or any other modified base capable of hybridization, either at the sugar level, for example the replacement of at least one deoxyribose with a poly
- information sequence means any ordered sequence of nucleotide type patterns, the chemical nature and order in a reference direction constitute information analogous to that given by the sequence of natural nucleic acids;
- hybridization means the process during which, under appropriate conditions, two nucleotide fragments having sufficiently complementary sequences are capable of associating by stable and specific hydrogen bonds, to form a double strand; the hybridization conditions are determined by "stringency", that is to say the rigor of the operating conditions; hybridization is all the more more specific than it is performed at higher stringency; the stringency is a function in particular of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids; the stringency can also be a function of the parameters of the hybridization reaction, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the temperature of hybridization; the stringency of the conditions under which a hybridization reaction must be carried out depends in particular on the probes used; all these data are well known and the appropriate conditions can possibly be determined in each case by routine experiments; in general, depending on the length of the probes used, the conditions of high string
- a “probe” is a nucleotide fragment comprising for example from 1 2 to 1 00 nucleotide units, in particular from 1 2 to 35 nucleotide units, having a specificity of hybridization under determined conditions to form a hybridization complex with an acid target nucleic acid comprising, in the present case, a nucleotide sequence derived from the origin of replication of the vector pBR322; a probe can in particular be used for diagnostic purposes, in the form in particular of a capture or detection probe;
- a “capture probe” is immobilized or immobilizable on a solid support by any suitable means, for example by covalence, by adsorption, or by direct synthesis on a solid support; the latter is presented in any suitable form such as tube, cone, well, microtiter plate, sheet, soluble polymer; it consists of a natural, synthetic material, chemically modified or not and is, according to the technique chosen, chosen from polystyrenes, styrene-butadiene copolymers, styrene-butadiene copolymers in admixture with polystyrenes, polypropylenes, polycarbonates , polystyrene-acrylonitrile copolymers, styrene-methylmethacrylate copolymers methyl, among nylon and natural synthetic fibers, among polysaccharides and cellulose derivatives;
- a “detection probe” can be marked by means of a marker chosen for example from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorigenic or luminescent substrate (in particular a peroxidase or a phosphatase alkaline), chromophoric chemicals, chromogenic, fluorigenic or luminescent compounds, nucleotide base analogs, and ligands such as biotin;
- a "primer” is a probe comprising for example from 1 2 to 1 00 nucleotide units and having a specificity of hybridization under conditions determined for the initiation of an enzymatic polymerization, for example in an amplification technique.
- the probes according to the invention are used for diagnostic purposes, in the search for the presence or absence of a target nucleotide sequence in a sample, according to all the known hybridization techniques and in particular the point deposition techniques on filter, say “DOT-BLOT” (MANIATIS et al, Moiecular Cloning, Cold Spring Harbor, 1 982), DNA transfer techniques called “SOUTHERN BLOT” [SOUTHERN. E.M., J. Mol.
- the sandwich technique is advantageously used, with at least one capture probe and / or at least one detection probe; in the case where the two types of probes are used, at least one of said probes (generally the detection probe) is capable of hybridizing with a region of the target which is specific for the sought sequence, it being understood that the capture probe and the detection probe must have nucleotide sequences at least partially different so that said probes are capable of hybridizing with two different regions of the target nucleic acid.
- the main stages of the so-called “sandwich” technique consist first of immobilizing a capture probe on a support, in particular by passive or covalent adsorption, bringing the immobilized probe (s) into contact with a sample, optionally pretreated, capable of containing at least one nucleotide sequence to be detected, called the target sequence, under conditions allowing hybridization of the or probes with a target sequence complementary to said probe (s), and detecting the hybridization complex formed therebetween.
- the detection of the complex is carried out by bringing it into contact with a detection probe, labeled with a labeling agent, then direct or indirect detection in particular by revealing the labeling agent.
- the detection probe can of course be present from the first step of the “sandwich” protocol.
- the detection probe is not marked.
- the detection of the target probe / nucleic acid complex can be achieved by the use of specific detection means for nucleic acids in their double-stranded form.
- specific detection means for nucleic acids for nucleic acids in their double-stranded form.
- a fragment of the invention comprises at least twelve nucleotide motifs capable of hybridizing to a region of SEQID NO: 1 or of its complement, under conditions of high stringency defined above.
- Excluded from the invention are the fragments whose sequence is chosen from the sequence of the plasmid pBR322 available in GenBank under the access number J01 749 and SEQ ID NO: 44 to 47.
- a preferred fragment of the invention comprises, or consists of, at least twelve contiguous nucleotide motifs belonging to SEQ ID NO: 1, and more advantageously still belonging to a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 43 and the complementary ones from SEQ ID NO: 3 to SEQ ID NO: 43.
- a second object of the invention is a nucleotide probe for the capture and / or detection of a gene therapy vector or of degradation products of such a vector, and more particularly of a nucleotide sequence derived from the origin of replication of pBR322, which comprises or consists of a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, under conditions of high stringency, to SEQ ID NO: 1 or its complement, and preferably in a fragment comprising or consisting of a sequence chosen from SEQ ID NO: 1 and SEQ ID NO: 3 to SEQ ID NO: 43, and their complement.
- a probe for the specific detection of a said nucleotide sequence is advantageously chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complementary ones from SEQ ID NO: 23 to SEQ ID NO: 43.
- capture probes have been defined according to the invention which preferably comprise or consist of a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 22 and the complementary ones of SEQ ID NO: 3 to SEQ ID NO: 22, and the detection probes which preferably comprise or consist of a sequence chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complementary ones of SEQ ID NO: 23 to SEQ ID NO: 43.
- Another object of the invention is a primer for the enzymatic amplification, for example according to the principle of PCR, of at least one nucleotide sequence derived from the origin of replication of pBR322, characterized in that it comprises or it consists of a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, under conditions of high stringency, to SEQ ID NO: 1 or to its complement, and preferably, a fragment comprising or consisting of a sequence chosen from SEQ ID NO: 1 and SEQ ID NO: 3 to SEQ ID NO: 43, and their complementary.
- it comprises or consists of a sequence chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complementary ones from SEQ ID NO: 23 to SEQ ID NO: 43.
- the invention also relates to a reagent for detecting and / or identifying and / or quantifying a nucleotide sequence derived from the origin of replication of pBR322 or a vector and / or a vector fragment comprising such a nucleotide sequence, comprising at least one probe. capture and at least one detection probe as defined above, and optionally at least one primer of the invention.
- Another subject of the invention is a method for detecting and / or identifying and / or quantifying a nucleotide sequence derived from the origin of replication of pBR322 or a vector and / or a vector fragment comprising such a nucleotide sequence, in a sample biological, capable of containing at least one said nucleotide sequence. It includes the steps of:
- the detection method is advantageously a technique
- said sample is brought into contact with a first probe, namely a capture probe of the invention, and a second probe, namely a detection probe of the invention. It advantageously comprises a preliminary step of amplification of said nucleotide sequence, which is preferably carried out by bringing the sample into contact with a primer described above.
- Step (b) can be carried out by means of an antibody, optionally coupled to a marker agent, directed against said hybridization complex.
- the invention also relates to the use of a probe and / or a primer previously described, for determining the presence or absence of a nucleotide sequence derived from the origin of replication of pBR322 or a vector and / or a vector fragment comprising such a nucleotide sequence in a biological sample.
- An appropriate biological sample is chosen in particular from a sample, of the fluid type (such as blood, urine), tissue or tissue fragment, mucus, organ or organ fragment, or culture supernatant obtained using one of the aforementioned direct debits.
- such a biological sample comes from a patient previously treated by a gene therapy protocol using a vector as defined according to the present invention.
- a sample consists of a blood sample but it is preferred in the context of the present invention to take samples of pulmonary or muscular origin. More specifically, such a sample is taken from the site of administration of the vector during the implementation of the gene therapy protocol.
- the use according to the invention can also relate to the study of the dissemination of a vector derived from pBR322, in particular in the patient's organism, in the environment or even in biological products originating from a patient treated with genetical therapy.
- the use according to the invention will make it possible to reliably determine whether the biological material donated by the patient contains or not a vector. derived from pBR322.
- the use according to the invention thus responds to the ever increasing need for security when donating biological material.
- GMOs genetically modified organisms
- the invention is illustrated by the following example of detection of a vector derived from pBR322 by the “sandwich” technique, after amplification.
- 293 cells are transfected with the plasmid pCH 1 04 (Hall et al., J. of Molecular and Applied Genetics, 1 983, 2, 1 01 -1 09) whose origin of replication is derived from pBR322 using the Calcium Phosphate Transfection System kit from GIBCO-BRL following the manufacturer's instructions.
- the plamid DNA is extracted from the cells by the HIRT protocol, in which the cells are lysed with the following buffer: 24 ⁇ l of SDS 1 0%
- Phenol / chloroform extraction is then carried out followed by precipitation with 100% ethanol. After centrifugation, the plasmid DNA pellet is taken up in an RB buffer. • PCR amplification
- the primers used for the amplification reaction include the following sequences:
- the amplification reaction is carried out under the conditions detailed below.
- the total volume of the reaction is 1 00 ⁇ l. It comprises a 50 mM Tris-HCI buffer, pH 8.5, containing 4 mM of MgCl 2 , 1,00 ⁇ g / ml of BSA (bovine serum albumin), 1 ⁇ M of each primer, 200 ⁇ M of each dNTP and 1 U of Taq polymerase.
- the reaction medium is then subjected to a first series of 5 cycles then to a second series of 30 amplification cycles.
- the DNA is first denatured by incubating the samples at 94 ° C for 10 seconds, then at 55 ° C for 10 seconds, so that the primers can pair with the template DNA, and finally at 72 ° C for 30 seconds to allow extension of the primers.
- the cycles are as follows: 94 ° C for 10 seconds, then 60 ° C for 10 seconds and 72 ° C for 30 seconds.
- the capture and detection probes are chosen so as to be complementary to two non-overlapping regions in the amplified DNA nucleotide sequence.
- the capture probe chosen is SEQ ID: 10 and the detection probe SEQ ID NO: 32
- the latter is linked to an enzymatic peroxidase group which will make it possible to demonstrate the formation of the hybridization complexes. This marking is carried out according to the procedure described in PCR protocols: A guide to methods and application; Press Academy (1,990), 1 5, p451 3-4534.
- the enzymatic activity is revealed by colorimetry.
- the capture probe is fixed to the walls of the wells of the microplate. This can be done by adsorption (Cook et al, NAR, 1 6, 4077-4095, 1 988) or by covalent coupling (Rasmussen, et al, 1 991, Analytical Biochemistry, 1 98, 1 38-142).
- the amplification reaction product is denatured in an NaOH / EDTA solution, then introduced by means of a hybridization buffer solution into the wells of the microplate at the same time as the detection probe.
- a hybridization buffer solution 100 mM Tris, 3M NaCl, 1% Tween 20, pH 7.4
- the peroxidase activity linked to the detection probe is detected by colorimetry. This detection is carried out using a solution of trisodium citrate and orthophenylenediamine (OPD).
- OPD orthophenylenediamine
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Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU31717/00A AU3171700A (en) | 1999-03-05 | 2000-03-03 | Nucleotide fragment, probe, primer, reagent and method for detecting a nucleotide sequence derived from pbr322 replication origin |
JP2000603424A JP2002537856A (ja) | 1999-03-05 | 2000-03-03 | ヌクレオチド断片、プローブ、プライマー、試薬、及びpBR322の複製起点由来のヌクレオチド配列を検出する方法 |
CA002366108A CA2366108A1 (fr) | 1999-03-05 | 2000-03-03 | Fragment nucleotidique, sonde, amorce, reactif et procede de detection d'une sequence nucleotidique derivee de l'origine de replication de pbr322 |
EP00909424A EP1159452A1 (fr) | 1999-03-05 | 2000-03-03 | FRAGMENT NUCLEOTIDIQUE, SONDE, AMORCE, REACTIF ET PROCEDE DE DETECTION D'UNE SEQUENCE NUCLEOTIDIQUE DERIVEE DE L'ORIGINE DE REPLICATION DE pBR322 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR99/02968 | 1999-03-05 | ||
FR9902968 | 1999-03-05 |
Publications (1)
Publication Number | Publication Date |
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WO2000053803A1 true WO2000053803A1 (fr) | 2000-09-14 |
Family
ID=9543028
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR2000/000543 WO2000053803A1 (fr) | 1999-03-05 | 2000-03-03 | FRAGMENT NUCLEOTIDIQUE, SONDE, AMORCE, REACTIF ET PROCEDE DE DETECTION D'UNE SEQUENCE NUCLEOTIDIQUE DERIVEE DE L'ORIGINE DE REPLICATION DE pBR322 |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1159452A1 (fr) |
JP (1) | JP2002537856A (fr) |
AU (1) | AU3171700A (fr) |
CA (1) | CA2366108A1 (fr) |
WO (1) | WO2000053803A1 (fr) |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0408918A1 (fr) * | 1989-06-23 | 1991-01-23 | Canon Kabushiki Kaisha | Méthode de détection d'acide nucléique |
US5212087A (en) * | 1988-01-28 | 1993-05-18 | Institut National De La Recherche Agronomique (Inra) | Ars sequence which is efficacious in yarrowia lipolytica |
WO1995015392A1 (fr) * | 1993-11-30 | 1995-06-08 | E.I. Du Pont De Nemours And Company | Genes chimeres et procedes d'augmentation de la teneur en lysine de semences de ble, de soja et de colza |
WO1996033207A1 (fr) * | 1995-04-18 | 1996-10-24 | Glaxo Group Limited | Amplification en chaine par polymerase a extremite complementaire |
US5599675A (en) * | 1994-04-04 | 1997-02-04 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
DE19718705A1 (de) * | 1997-05-02 | 1998-11-05 | Christoph Dr Fiehn | Verfahren zur quantitativen Bestimmung der Anzahl von Transgenkopien in gentechnisch veränderten Zellen |
US5840851A (en) * | 1993-07-23 | 1998-11-24 | Plomer; J. Jeffrey | Purification of hemoglobin |
WO1999006072A1 (fr) * | 1997-07-30 | 1999-02-11 | Boehringer Mannheim Corporation | Promedicaments cyclises |
WO1999014318A1 (fr) * | 1997-09-16 | 1999-03-25 | Board Of Regents, The University Of Texas System | Synthese chimique complete et synthese de genes et de genomes |
-
2000
- 2000-03-03 WO PCT/FR2000/000543 patent/WO2000053803A1/fr not_active Application Discontinuation
- 2000-03-03 JP JP2000603424A patent/JP2002537856A/ja not_active Withdrawn
- 2000-03-03 CA CA002366108A patent/CA2366108A1/fr not_active Abandoned
- 2000-03-03 AU AU31717/00A patent/AU3171700A/en not_active Abandoned
- 2000-03-03 EP EP00909424A patent/EP1159452A1/fr not_active Withdrawn
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5212087A (en) * | 1988-01-28 | 1993-05-18 | Institut National De La Recherche Agronomique (Inra) | Ars sequence which is efficacious in yarrowia lipolytica |
EP0408918A1 (fr) * | 1989-06-23 | 1991-01-23 | Canon Kabushiki Kaisha | Méthode de détection d'acide nucléique |
US5840851A (en) * | 1993-07-23 | 1998-11-24 | Plomer; J. Jeffrey | Purification of hemoglobin |
WO1995015392A1 (fr) * | 1993-11-30 | 1995-06-08 | E.I. Du Pont De Nemours And Company | Genes chimeres et procedes d'augmentation de la teneur en lysine de semences de ble, de soja et de colza |
US5599675A (en) * | 1994-04-04 | 1997-02-04 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
WO1996033207A1 (fr) * | 1995-04-18 | 1996-10-24 | Glaxo Group Limited | Amplification en chaine par polymerase a extremite complementaire |
DE19718705A1 (de) * | 1997-05-02 | 1998-11-05 | Christoph Dr Fiehn | Verfahren zur quantitativen Bestimmung der Anzahl von Transgenkopien in gentechnisch veränderten Zellen |
WO1999006072A1 (fr) * | 1997-07-30 | 1999-02-11 | Boehringer Mannheim Corporation | Promedicaments cyclises |
WO1999014318A1 (fr) * | 1997-09-16 | 1999-03-25 | Board Of Regents, The University Of Texas System | Synthese chimique complete et synthese de genes et de genomes |
Non-Patent Citations (1)
Title |
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DATABASE EMSYN EMBL; XP002124483 * |
Also Published As
Publication number | Publication date |
---|---|
AU3171700A (en) | 2000-09-28 |
EP1159452A1 (fr) | 2001-12-05 |
JP2002537856A (ja) | 2002-11-12 |
CA2366108A1 (fr) | 2000-09-14 |
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