AU3171700A - Nucleotide fragment, probe, primer, reagent and method for detecting a nucleotide sequence derived from pbr322 replication origin - Google Patents
Nucleotide fragment, probe, primer, reagent and method for detecting a nucleotide sequence derived from pbr322 replication origin Download PDFInfo
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Description
WO 00/53803 1 PCT/FROO/00543 NUCLEOTIDE FRAGMENT, PROBE, PRIMER, REAGENT AND METHOD FOR DETECTING A NUCLEOTIDE SEQUENCE DERIVED FROM THE ORIGIN OF REPLICATION OF pBR322 5 The present invention relates to the detection, using nucleotide probes, of nucleotide sequences derived from the origin of replication of the pBR322 vector, which are present in gene therapy vectors. Gene therapy is defined as the transfer of genetic information of 10 therapeutic interest into a host cell or organism. The first protocol applied to humans was initiated in the United States in September 1990 on a patient who was genetically immuno deficient due to a mutation affecting the gene encoding adenine deaminase (ADA). It involves correcting or replacing the defective gene, the dysfunction of which is the cause of a genetic disease with a functional gene. 15 The relative success of this first experiment has encouraged the development of this technology which has since been extended to the treatment of other diseases, both genetic and acquired (cancers, infectious diseases such as AIDS, etc), with the aim of delivering therapeutic genes in situ. Most strategies use vectors for vehiculing the therapeutic gene towards its cellular target. Many 20 vectors, both viral and synthetic, have been developed over the last few years and have been the subject of many publications which are accessible to those skilled in the art (see, for example, Robbins et al, 1988, Tibtech, 16, 34-40 and Rolland, 1998, Therapeutic Drug Carrier Systems, 15, 143-198). In the context of monitoring a gene therapy in a patient to whom a 25 medicinal product has been administered in the form of a vector carrying a gene of therapeutic interest, it is important to survey various parameters and in particular the following parameters: the presence or absence of the vector, or even of its degradation products, and its location. The traceability of the vector in fact has many advantages: 30 - clinical advantages, since the demonstration of the disappearance of the vector may lead the physician to adjust the treatment and to prescribe a new administration of said vector, - advantages with respect to the regulations and ethical advantages, since it allows the in vivo evolution of the medicinal vector product 35 to be monitored.
WO 00/53803 2 PCT/FROO/00543 It is therefore advisable to have tests for detecting the presence of the vector administered or at least of fragments thereof. Currently, diagnostic tests are carried out by certain laboratories associated with the place where treatment takes place and by biotechnology 5 service companies, according to protocols which are not standardized, which are neither specific nor optimized, which do not satisfy conditions of good laboratory practice and which produce relatively non reproducible results which do not permit their systematic use in the context of monitoring a gene therapy treatment or, more particularly, in the cases of controls for in vivo and ex vivo 10 dissemination of vectors or in the cases of preclinical and clinical studies. In order to satisfy the need for a test which does not have the abovementioned drawbacks, the aim of the present invention is to provide a sensitive, reliable detection test which can be applied to a large number of vectors used in gene therapy protocols. 15 The pBR322 plasmid (Genbank accession number 27-4902-01), derived from the ColE1 wild-type plasmid, is commonly used as starting material for preparing 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 novel vectors [F. Bolivar et al, Gene 20 2, 95 (1977) and N. Watson, Gene, 70, 399 (1988)]. In particular, the region of pBR322 comprising its origin of replication has been used as a basis for constructing many gene therapy vectors. According to the invention, a tool and a method for detecting a 25 nucleotide sequence derived from the origin of replication of said vector, which can be applied to any vector derived from pBR322, are provided. Since the ColE1 plasmid, from which pBR322 is derived, is omnipresent in humans, it is also essential to be able to distinguish the modified form of the origin of replication, which is characteristic of the presence 30 of a vector comprising the origin of replication of pBR322, from the wild-type form of the origin of replication, in order to obtain a specific test. Specificity is another advantage of the test provided according to the invention. Thus, a first subject of the invention is a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide units 35 capable of hybridizing, under conditions of high stringency, to the sequence defined by SEQ ID No : 1 or to the sequence complementary thereto.
WO 00/53803 3 PCT/FROO/00543 Preferably, said sequence comprises at least twelve contiguous nucleotide units of a sequence chosen from SEQ ID No : 1 and a sequence complementary thereto. In the context of the present invention, such a fragment preferably constitutes a probe. 5 The sequence SEQ ID No : 1 described at the end of the description in the sequence listing corresponds to the complementary DNA sequence beginning at nucleotide 2140 and ending at nucleotide 3145 of the sequence of the plasmid available in the data banks (GenBank, www.ncbi.nlm.nih.gov/) under the accession number J01749, and SEQ ID No : 10 2 corresponds to that of the ColE1 plasmid, for the same region (accession number J01 566 in the same bank). Before explaining the invention in greater detail, various terms used in the description and claims are hereinafter defined: - the term ( vector derived from pBR322 , is intended to denote the 15 recombinant nucleic acid constructs in which at least the origin of replication is derived from pBR322. According to a particular case of the invention, such a vector will consist of a plasmid, it being understood, moreover, that said vector may also comprise nucleic acid sequences heterologous to pBR322 and in particular nucleic acid sequences or nucleic acid constructs having a 20 therapeutic interest. Advantageously, the gene of therapeutic interest encodes an antisense RNA, a ribozyme or a polypeptide of interest. It may be derived from a eukaryotic organism, from a procaryot, from a parasite or from a virus. It may be isolated by any conventional technique in the field of the art (by cloning, PCR, 25 chemical synthesis), etc. It may be of genomic type (comprising all or part of the set of introns), of complementary DNA type (cDNA, lacking introns) or of mixed type (minigene). Moreover, the polypeptide for which it codes may be (i) intracellular, (ii) incorporated into the membrane of the host cell or (iii) secreted. It may be a polypeptide as found naturally (native), a portion thereof (truncated), 30 a mutant having in particular improved or modified biological properties, or a chimeric polypeptide originating from the fusion of sequences of diverse origins. Among the polypeptides of interest which may be used, mention may more particularly be made of chemokines (MIP-1a, MIP-1b, RANTES, DC-CKI, MDC, MCP1 (monocyte chemoattractant protein), IP10, etc.), cytokines (a-, b- or 35 g-interferon, interleukin (IL), in particular IL-2, IL-6, IL-10 or IL-12, colony stimulating factor (GM-CSF, C-CSF, M-CSF), etc.), cell receptors (in particular WO 00/53803 4 PCT/FROO/00543 recognized by the HIV virus), receptor ligands, clotting factors (Factor Vill, Factor IX, thrombin, protein C), growth factors, proangiogenic factors (FGF for Fibroblast Growth Factor, VEGF for Vascular Endothelial Growth Factor, SH[sic]/HGF for scatter factor/hepatocyte growth factor, TGF for transforming 5 growth factor, TNF for tumor necrosis factor, angiopoitin), enzymes (urease, renin, metalloprotinase, nitric oxide synthetase NOS, SOD, catalase, lecithin cholesterol acyltransferase LCAT, etc.), enzyme inhibitors (al-antitrypsine, antithrombin III, viral protease inhibitor, PAl-1 for plasminogen activator inhibitor), class I or Il major histocompatibility complex anitigens or polypeptides which act 10 on the expression of the corresponding genes, antigens (or antigenic peptides) capable of generating an immune response, polypeptides capable of inhibiting a viral, bacterial or parasitic infection or the development thereof, polypeptides with antitumor effect (tumor suppressor gene expression products, tumor-associated antigens, etc.), polypeptides which act positively or negatively on apoptosis (Bax, 15 Bcl2, BcIX, etc.), cytostatic agents (p21, p16, Rb), whole immunoglobulins or part immunoglobulins (Fab, ScFv...), toxins, immunotoxins, apolipoproteins (ApoAl, ApoAIV, ApoE, etc.), cytotoxic products, anti-angiogenic factors (angiostatin, endostatin, PF-4, etc.), markers (b-galactosidase, luciferase, green fluorescent protein) or any other polypeptide having a therapeutic effect on the condition 20 targeted. More precisely, with the aim of treating a hereditary dysfunction, a functional copy of the defective gene will be used, for example a gene encoding factor Vill or IX in the context of hemophilia A or B, dystrophin (or minidystrophin) in the context of Duchenne and Becker myopathies, insulin in the context of 25 diabetes and the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein in the context of cystic fibrosis. As regards inhibiting the initiation or progression of tumors or cancers, use may preferably be made of a gene of interest encoding an antisense RNA, a ribozyme, a cytotoxic product (thymidine kinase of herpes simplex virus 1 (TK-HSV-1), ricin, cholera toxin, 30 diptheria toxin, product of the yeast genes FCY1 and FUR1 encoding uracil phosphoribosyl transferase and cytosine deaminase), an immunoglobulin, an inhibitor of cell division or of transduction signals, a tumor suppressor gene expression product (p53, Rb, p73, DCC, etc.), a polypeptide which stimulates the immune system, or a tumor-associated antigen (MUC-1, BRCA-1, early or late 35 antigens of a papilloma virus), optionally in combination with a cytokine gene. Finally, in the context of anti-HIV therapy, use may be made of a gene encoding WO 00/53803 5 PCT/FROO/00543 an immunoprotective polypeptide, an antigenic epitope, an antibody (2F5; Buchacher et al., 1992, Vaccines 92, 191-195), the extracellular domain of the CD4 receptor (sCD4 ; Traunecker et al., 1988, Nature 331, 84-86), an immunoadhesion (for example a CD4-IgG immunoglobulin hybrid; Capon et al., 5 1989, Nature 337, 525-531 ; Byrn et al., 1990, Nature 344, 667-670), an immunotoxin (for example fusion of the 2F5 antibody or of the CD4-2F5 immunoadhesin to angiogenin; Kurachi et al., 1985, Biochemistry 24, 5494 5499), a transdominant variant (EP 0614980, W095/16780), a cytotoxic product such as one of those mentioned above, or an EIFN or IIFN. 10 According to the invention, said vectors or nucleic acid constructs which may be used in gene therapy may be in their naked form (Wolff et al., 1990, Science 2, page 1465-1468.), combined with liposomes, cationic lipids, cationic polymers, peptides or polypeptides. The literature relating to the vectors which may be used in gene therapy provides a considerable number of 15 examples of such vectors (see, for example, Robbins et al, 1988, Tibtech, 16, 34-40 and Rolland, 1998, Therapeutic Drug Carrier Systems, 15, 143-198). - the term "nucleotide sequence" is intended to mean a linear or circular, single-stranded or double-stranded DNA sequence; - the terms "nucleotide fragment" and "oligonucleotide" are two 20 synonymous terms which denote a chain of nucleotide units, characterized by the informational sequence of the nucleic acids which are natural (or optionally modified) 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 25 structure which is different from that of natural nucleic acids; a nucleotide (or oligonucleotide) fragment generally contains at least 12 nucleotide units and may be obtained from a natural nucleic acid molecule and/or by genetic recombination and/or by chemical synthesis; - a nucleotide unit is derived from a monomer which may be a 30 natural nucleotide of nucleic acid in which the constituent elements are a sugar, a phosphate group and a nitrogenous base; in DNA, the sugar is 2-deoxy ribose, and in RNA, the sugar is ribose; depending on whether it is a question of DNA or RNA, the nitrogenous base is chosen from adenine, guaninine, uracil, cytosine and thymine; or alternatively the monomer is a nucleotide 35 modified in at least one of the three constituent elements; by way of example, the modification may occur either in the bases, with modified bases such as WO 00/53803 6 PCT/FROO/00543 inosine, 5-methyldeoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine or any other modified base capable of hybridization, in the sugar, for example replacement of at least one of deoxyribose with a polyamide [P.E. Nielsen et al, Science, 254, 1497-1500 5 (1991)], or even in the phosphate group, for example replacement thereof with esters chosen in particular from diphosphates, alkyl- and arylphosphonates, and phosphorothioates; - the term "informational sequence" is intended to mean any ordered series of units of nucleotide type, in which the chemical nature and 10 order in a reference direction constitute information analogous to that given by the sequence of the natural nucleic acids; - the term "hybridization" is intended to mean the process during which, under suitable conditions, two nucleotide fragments having sufficiently complementary sequences are capable of associating via stable and specific 15 hydrogen bonds so as to form a double strand; the hybridization conditions are determined by the "stringency", i.e. the severity of the operating conditions; the higher the stringency at which the hybridization is carried out, the more specific it is; the stringency depends, in particular, on the base composition of a probe/target duplex and also by [sic] the degree of mismatching between two 20 nucleic acids; the stringency may also depend on the parameters of the hybridization reaction, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents and/or the hybridization temperature; the stringency of the conditions under which a hybridization reaction must be carried out depends in particular 25 on the probes used; all these data are well known and the suitable conditions may optionally be determined in each case using routine experiments; in general, depending on the length of the probes used, conditions of high stringency are as follows: the hybridization reaction is carried out at a temperature of between approximately 20 and 65 0 C, in particular between 35 30 and 65 *C, in a saline solution at a concentration of approximately 0.3 to 1 M; in particular, under the hybridization conditions according to the present invention, at a temperature of 37 0 C + 1 C, in a 3 x PBS saline solution (0.45 M NaCl; 0.15 M sodium phosphate), is chosen; - a "probe" is a nucleotide fragment comprising, for example, from 35 12 to 100 nucleotide units, in particular from 12 to 35 nucleotide units, and having hybridization specificity under given conditions for forming a WO 00/53803 7 PCT/FROO/00543 hybridization complex with a target nucleic acid comprising, in the present case, a nucleotide sequence derived from the origin of replication of the pBR322 vector; a probe may in particular be used for diagnostic purposes, especially in the form of a capture or detection probe; 5 - a "capture probe" is or can be immobilized on a solid support by any suitable means, for example by covalents, by adsorption, or by direct synthesis on a solid support; the latter is in any suitable form, such as a tube, cone, well, microtitration plate, sheet or soluble polymer; it consists of a natural or synthetic material, which may or may not be chemically modified, and is, 10 depending on the technique selected, chosen from polystyrenes, styrene/butadiene copolymers, styrene/butadiene copolymers mixed with polystyrenes, polypropylenes, polycarbonates, polystyrene/acrylonitrile copolymers and styrene/methyl methyl[sic] methacrylate copolymers, from synthetic nylon fibers and natural fibers, and from polysaccharides and 15 cellulose derivatives. - a "detection probe" may be labeled using a label chosen, for example, from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorogenic or luminescent substrate (in particular a peroxydase or an alkaline phosphatase), the chromophoric chemical 20 compounds, chromogenic, fluorogenic or luminescent compounds, nucleotide base analogs and ligands such as biotin; - a "primer" is a probe comprising, for example, from 12 to 100 nucleotide units and having hybridization specificity under given conditions for initiating enzymatic polymerization, for example in an amplification technique. 25 The probes according to the invention are used for diagnostic purposes, in searching for the presence or absence of a target nucleotide sequence in a sample, according to any known hybridization techniques and in particular "DOT-BLOT" techniques (MANIATIS et al, Molecular Cloning, Cold Spring Harbor, 1982), "SOUTHERN BLOT" DNA transfer techniques 30 [SOUTHERN. E.M., J. Mol. Biol., 98, 503 (1975)], or "sandwich" techniques [DUNN A.R., HASSEL J.A., Cell, 12, 23 (1977)] ; the sandwich technique is advantageously used, with at least one capture probe and/or at least one detection probe; when two types of probe are used, at least one of said probes (generally the detection probe) is capable of hybridizing with a region of the 35 target which is specific for the sequence being sought, it being understood that the capture probe and the detection probe must have nucleotide sequences WO 00/53803 8 PCT/FROO/00543 which are at least partially different so that said probes are capable of hybridizing with two different regions of the target nucleic acid. The main steps of the "sandwich" technique consist, first, in immobilizing a capture probe on a support, in particular by passive adsorption 5 or covalence, in bringing the immobilized probe(s) into contact with an optionally pretreated sample likely to contain at least one nucleotide sequence to be detected, termed target sequence, under conditions which allow the hybridization of the probe(s) with a target sequence complementary to said probe(s) and in detecting the hybridization complex formed between the latter. 10 The complexe is detected by bringing it into contact with a detection probe, labeled with a labeling agent, and then detecting it directly or indirectly, in particular by revealing the labeling agent. The detection probe may, of course, be present from the first step of the "sandwich" protocol. According to one particular case of the invention, the detection 15 probe is not labeled. In this case, the probe/target nucleic acid complex may be detected using detection means which are specific for nucleic acids in their double-stranded form. To this effect and by way of example, it is possible to use antibodies capable of recognizing the double-stranded structures of nucleic acids (WO-A-97/32602). 20 The various subjects of the invention and their preferred variants are set out hereinafter. As mentioned above, a fragment of the invention comprises at least twelve nucleotide units capable of hybridizing to a region of SEQ ID No: 1 or of the sequence complementary thereto, under conditions of high stringency 25 defined above. Excluded from the invention are the fragments for which the sequence is chosen from the sequence of the pBR322 plasmid available in GenBank under the accession number J01 749 and SEQ ID No :44 to 47. Since the nucleotide sequences derived from the origin of 30 replication of pBR322 are well conserved, a preferred fragment of the invention comprises, or consists of, at least twelve contiguous nucleotide units belonging to SEQ ID No : 1, and even more advantageously belonging to a sequence chosen from SEQ ID No : 3 to SEQ ID No : 43 and the sequences complementary to SEQ ID No: 3 to SEQ ID No: 43. 35 A second subject of the invention is a nucleotide probe for capturing and/or detecting a gene therapy vector or degradation products of WO 00/53803 9 PCT/FRO0/00543 such a vector, and more particularly a nucleotide sequence derived from the origin of replication of pBR 322, which comprises, or which consists of, a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide units capable of hybridizing, under conditions of high 5 stringency, to SEQ ID No : 1 or to a sequence complementary thereto, and preferably of 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 the sequences complementary thereto. A probe for the specific detection of a said nucleotide sequence is advantageously chosen from SEQ ID No : 23 to SEQ ID No : 43 10 and the sequences complementary to SEQ ID No: 23 to SEQ ID No: 43. For implementing the "sandwich" detection technique in particular, capture probes which preferably comprise, or consist of, a sequence chosen from SEQ ID No : 3 to SEQ ID No : 22 and the sequences complementary to SEQ ID No : 3 to SEQ ID No : 22 and detection probes which preferably 15 comprise, or consist of, a sequence chosen from SEQ ID No : 23 to SEQ ID No: 43 and sequences complementary to SEQ ID No: 23 to SEQ ID No : 43, have been defined according to the invention. Another subject of the invention is a primer for enzymatically amplifying, for example according to the PCR principle, at least one nucleotide 20 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 units capable of hybridizing, under conditions of high stringency, to SEQ ID No : 1 or to the sequence complementary thereto, and preferably of a fragment comprising, or 25 consisting of, a sequence chosen from SEQ ID No : 1 and SEQ ID NO : 3 to SEQ ID No : 43, and the sequences complementary thereto. In particular, it comprises, or consists of, a sequence chosen from SEQ ID No : 23 to SEQ ID No : 43 and the sequences complementary to SEQ ID No: 23 to SEQ ID No: 43. 30 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 capture probe and at least one detection probe as defined above and, optionally, at least one primer of the 35 invention.
WO 00/53803 10 PCT/FROO/00543 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 biological sample likely to contain at least one said 5 nucleotide sequence. It comprises the steps consisting: (a) in bringing said sample into contact with at least one probe of the invention, under conditions which allow the formation of a hybridization complex, and (b) in detecting, by any suitable means, the formation of a 10 complex from hybridization between said probe and said nucleotide sequence. The detection method is advantageously a "sandwich" technique. In this particular case, said sample is brought into contact with a first probe, namely a capture probe of the invention, and a second probe, namely a 15 detection probe of the invention. It advantageously comprises a preliminary step for amplifying said nucleotide sequence, which is preferably carried out by bringing the sample into contact with a primer described above. Step (b) may be carried out using an antibody which is optionally coupled to a labeling agent and which is directed against said hybridization 20 complex. The invention also relates to the use of a probe and/or of a primer described above, 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. 25 A suitable biological sample is in particular chosen from a specimen of the fluid (such as blood or urine), tissue or tissue fragment, mucous, organ or organ fragment type, or of the type of a culture supernatant obtained using one of the abovementioned specimens. Preferably, such a biological sample is derived from a patient pretreated with a gene therapy protocol using a vector as 30 defined according to the present invention. According to one particular case, such a sample consists of a blood specimen, but in the context of the present invention, samples of pulmonary or muscle origin are preferably taken. More specifically, such a specimen is taken at the site of administration of the vector during the implementation of the gene therapy protocol. 35 However, the use according to the invention may also relate to studying the dissemination of a vector derived from pBR322, in particular in the WO 00/53803 11 PCT/FROO/00543 patient's body, in the environment or in the biological products derived from a patient treated by gene therapy. In particular, when such a patient wishes to donate blood or one of his or her organs, the use according to the invention will make it possible to reliably determine whether or not the biological material 5 donated by the patient contains a vector derived from pBR322. The use according to the invention in this way satisfies the ever increasing need for safety when biological material is donated. It is also possible to envision applying the detection method of the invention to the agrofoods domain or to cosmetics. Specifically, these domains 10 increasingly turn to the use of genetic modified organisms (GMO), i.e. organisms transformed using a recombinant vector, for developing their products. The method of the invention therefore offers a reliable and effective means for determining whether a food or cosmetic product has been developed using a GMO. 15 The invention is illustrated by the following example of detection of a vector derived from pBR322 using the "sandwich" technique, after amplification. Example: Transfection of cells with the pCH104 plasmid 293-cells are transfected with the pCH104 plasmid (Hall et al., J. of 20 Molecular and Applied Genetics, 1983, 2, 101-109), the origin of replication of which is derived from pBR322, using the Calcium Phosphate Transfection System kit from GIBCO BRL, according to the manufacturer's indications. 0 DNA extraction The plasmid DNA is extracted from the cells using the HIRT 25 protocol, in which the cells are lysed with the following buffer: 24 01 of 10% SDS 8 01 of 0.5 M EDTA 8 El of 10 mg/m protinase K 160 El of TE, pH8 30 A 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 comprise the 35 following sequences: SEQ ID No :26 WO 00/53803 12 PCT/FROO/00543 SEQ ID No :38 The amplification reaction is carried out under the conditions detailed hereinafter. The total reaction volume is 100 pl. It comprises a 50 mM tris-HCI buffer, pH 8.5, containing 4 mM of MgCl 2 , 100 pg/ml of BSA (bovine 5 serum albumin), 1 pM of each primer, 200 pM of each dNTP and 1 U of taq polymerase. The reaction medium is then subjected to a first series of 5 application cycles and then a second series of 30 amplification cycles. In the first series, the DNA is first denatured by incubating the samples at 94 0 C for 10 seconds, then at 55*C for 10 seconds so that the primers may pair with the 10 DNA matrix, and finally at 72*C for 30 seconds in order to allow extension of the primers. In the second series, the cycles are as follows: 94 0 C for 10 seconds, 60 0 C for 10 seconds and 72*C for 30 seconds. 6 Detection on microplates The capture and detection probes are chosen so as to be 15 complementary to two nonoverlapping regions in the nucleotide sequence of DNA amplified. The capture probe chosen is SEQ ID No: 10 and the detection probe is SEQ ID No : 32. This latter is attached to a peroxidase enzymatic group which will make it possible to reveal the formation of the hybridization complexes. This labeling is carried out according to the procedure described in 20 PCR protocols: A guide to methods and application; Academic Press (1990), 15, p4513-4534. The enzymatic activity is revealed by colorimetry. The capture probe is attached to the walls of the microplate wells. This operation may take place by adsorption (Cook et al, NAR, 16, 4077-4095, 1988) or by covalent coupling (Rasmussen, et al, 1991, Analytical 25 Biochemistry, 198, 138-142). The product of the amplification reaction is denatured in a NaOH/EDTA solution and then introduced, by means of a hybridization buffer solution, into the wells of the microplate at the same time as the detection probe. 30 After an incubation phase (1 hour at 37 0 C with agitation) and a washing phase (100 mM tris, 3M NaCl, 1 % Tween 20, pH 7.4), the peroxydase activity attached to the detection probe is detected by colorimetry. This detection is carried out using a trisodium citrate and orthophenylenediamine (OPD) solution. After incubation for 30 minutes in the dark, a 4N H 2 S0 4 35 solution is added in order to stop the reaction. The optical density is then determined at 492 nm.
Claims (20)
1. A single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide units capable of 5 hybridizing, under conditions of high stringency, to SEQ ID No : 1 or to a sequence complementary thereto, excluding the fragments for which the sequence is chosen from the sequence of the pBR322 plasmid available in GenBank under the accession number J01 749 and SEQ ID No :44 to 47.
2. The fragment as claimed in claim 1, characterized in that 10 said sequence comprises at least twelve contiguous nucleotide units of a sequence chosen from SEQ ID No : 1 and the sequence complementary thereto.
3. The fragment as claimed in claim 2, characterized in that said sequence comprises at least twelve contiguous nucleotide units of a 15 sequence chosen from SEQ ID No : 3 to SEQ ID No : 43 and the sequences complementary to SEQ ID No: 3 to SEQ ID NO : 43.
4. The fragment as claimed in claim 2, characterized in that said sequence consists of a sequence chosen from SEQ ID No :3 to SEQ ID No :43 and the sequences complementary to SEQ ID No :3 to 20 SEQ ID No :43.
5. A probe for capturing and/or detecting a nucleotide sequence derived from the origin of replication of the pBR322 vector, characterized in that it comprises, or it consists of, a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous 25 nucleotide units capable of hybridizing, under conditions of high stringency, to SEQ ID No : 1 or to the sequence complementary thereto, and preferably of 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 the sequences complementary thereto. 30
6. A probe for capturing and/or detecting a vector and/or a vector fragment comprising a nucleic acid 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 units capable of hybridizing, under conditions 35 of high stringency, to SEQ ID No : 1 or to the sequence complementary thereto, and preferably of a fragment comprising, or consisting of, a WO 00/53803 14 PCT/FROO/00543 sequence chosen from SEQ ID No : 1 and SEQ ID No : 3 to SEQ ID No 43, and the sequences complementary thereto.
7. The probe as claimed in claim 5 and 6, characterized in that it is a capture probe and in that it comprises, or consists of, a sequence 5 chosen from SEQ ID No : 3 to SEQ ID No : 22 and the sequences complementary to SEQ ID NO: 3 to SEQ ID No : 22.
8. The probe as claimed in claim 7, characterized in that it is immobilized on a solid support.
9. The probe for specifically detecting a nucleotide sequence 10 derived from the origin of replication of pBR322 as claimed in claim 5, or a vector and/or a vector fragment as claimed in claim 6, characterized in that it comprises, or consists of, a sequence chosen from SEQ ID No :23 to SEQ ID No :43 and the sequences complementary to SEQ ID No :23 to SEQ ID No :43. 15
10. The probe as claimed in claim 9, characterized in that it is labeled with a labeling agent, in particular chosen from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorigenic or luminescent substrate, such as a peroxidase of alkaline phosphatase, chromophoric chemical compounds, chormogenic, fluorigenic 20 or luminescent compounds, nucleotide base analogs and ligands such as biotin.
11. A primer for enzymatically amplifying at least one nucleotide sequence derived from the origin of replication of the pBR322, characterized in that it comprises, or it consists of, a single-stranded 25 nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide units capable of hybridizing, under conditions of high stringency, to SEQ ID No : 1 or to the sequence complementary thereto, and preferably of 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 the sequences 30 complementary thereto.
12. The primer for specifically enzymatically amplifying at least one nucleotide sequence derived from the origin of replication of pBR322 as claimed in claim 11, characterized in that it comprises, or consists of, a sequence chosen from SEQ ID No: 23 to SEQ ID No: 43 and 35 the sequences complementary to SEQ ID No: 23 to SEQ ID No: 43. WO 00/53803 15 PCT/FROO/00543
13. 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 containing a said nucleotide sequence, characterized in that it comprises at least one capture probe as 5 claimed in claim 6 or 7 and at least one detection probe as claimed in claim 9 or 10.
14. The reagent as claimed in claim 13, characterized in that it also comprises a primer as claimed in claim 11 or 12.
15. A method for detecting and/or identifying and/or 10 quantifying a nucleotide sequence derived from the origin of replication of pBR322, or from a vector and/or a vector fragment containing a said nucleotide sequence, in a biological sample likely to contain at least one said nucleotide sequence, or one said vector and/or one said vector fragment, characterized in that it comprises the steps consisting: 15 (a) in bringing said sample into contact with at least one probe as claimed in any one of claims 5 to 10, under conditions which allow the formation of a hybridization complex, and (b) in detecting, by any suitable means, the formation of a complex from hybridization between said probe and said 20 nucleotide sequence.
16. The method as claimed in claim 15, characterized in that said sample is brought into contact with at least one first probe as claimed in claim 6 or 7 and at least one second probe as claimed in claim 9 or 10.
17. The method as claimed in claim 15 or 16, characterized in 25 that it comprises a preliminary step for amplifying said nucleotide sequence.
18. The method as claimed in claim 17, characterized in that said sample is brought into contact with at least one primer as claimed in claim 11 or 12.
19. The method as claimed in claim 15, characterized in that 30 step (b) is carried out using an antibody which is optionally coupled to a labeling agent and which is directed against said hybridization complex.
20. The use of a probe as claimed in any one of claims 5 to 10 and/or of a primer as claimed in claim 11 or 12, for determining the presence or absence of a nucleotide sequence derived from the origin of 35 replication of pBR322, of a vector and/or of vector fragment comprising a said nucleotide sequence, in a biological sample.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9902968 | 1999-03-05 | ||
FR9902968 | 1999-03-05 | ||
PCT/FR2000/000543 WO2000053803A1 (en) | 1999-03-05 | 2000-03-03 | NUCLEOTIDE FRAGMENT, PROBE, PRIMER, REAGENT AND METHOD FOR DETECTING A NUCLEOTIDE SEQUENCE DERIVED FROM pBR322 REPLICATION ORIGIN |
Publications (1)
Publication Number | Publication Date |
---|---|
AU3171700A true AU3171700A (en) | 2000-09-28 |
Family
ID=9543028
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU31717/00A Abandoned 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 |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1159452A1 (en) |
JP (1) | JP2002537856A (en) |
AU (1) | AU3171700A (en) |
CA (1) | CA2366108A1 (en) |
WO (1) | WO2000053803A1 (en) |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2626584B1 (en) * | 1988-01-28 | 1990-07-13 | Agronomique Inst Nat Rech | EFFECTIVE ARS SEQUENCE IN YARROWIA LIPOLYTICA AND PROCESS FOR ITS PREPARATION |
JP2802125B2 (en) * | 1989-06-23 | 1998-09-24 | キヤノン株式会社 | Nucleic acid detection method |
US5840851A (en) * | 1993-07-23 | 1998-11-24 | Plomer; J. Jeffrey | Purification of hemoglobin |
AU704510B2 (en) * | 1993-11-30 | 1999-04-22 | E.I. Du Pont De Nemours And Company | Chimeric genes and methods for increasing the lysine content of the seeds of corn, soybean and rapeseed plants |
US5834252A (en) * | 1995-04-18 | 1998-11-10 | Glaxo Group Limited | End-complementary polymerase reaction |
US5552278A (en) * | 1994-04-04 | 1996-09-03 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
DE19718705A1 (en) * | 1997-05-02 | 1998-11-05 | Christoph Dr Fiehn | Method for the quantitative determination of the number of transgene copies in genetically modified cells |
WO1999006072A1 (en) * | 1997-07-30 | 1999-02-11 | Boehringer Mannheim Corporation | Cyclized prodrugs |
DE69841578D1 (en) * | 1997-09-16 | 2010-05-06 | Centocor Inc | Methods for complete chemical synthesis and composition of genes and genomes |
-
2000
- 2000-03-03 WO PCT/FR2000/000543 patent/WO2000053803A1/en not_active Application Discontinuation
- 2000-03-03 JP JP2000603424A patent/JP2002537856A/en not_active Withdrawn
- 2000-03-03 AU AU31717/00A patent/AU3171700A/en not_active Abandoned
- 2000-03-03 EP EP00909424A patent/EP1159452A1/en not_active Withdrawn
- 2000-03-03 CA CA002366108A patent/CA2366108A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2000053803A1 (en) | 2000-09-14 |
CA2366108A1 (en) | 2000-09-14 |
JP2002537856A (en) | 2002-11-12 |
EP1159452A1 (en) | 2001-12-05 |
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