CA2366108A1 - Nucleotide fragment, probe, primer, reagent and method for detecting a nucleotide sequence derived from pbr322 replication origin - Google Patents

Abstract

The invention concerns a single-strand nucleotide fragment comprising a sequence of at least twelve adjacent nucleotide motifs capable of being hybridised, in conditions of high stringency, with SEQ ID NO: 1 or it complementary sequence. The invention also concerns the uses of said fragment as probe, primer, in a reagent and in a method for detecting a nucleotide sequence derived from pBR322 replication origin in a biological sample.

Description

NUCLEOTIDE FRAGMENT, PROBE, PRIMER, REAGENT AND METHOD
FOR DETECTION OF A NUCLEOTIDE SEQUENCE DERIVED FROM
THE ORIGIN OF REPLICATION OF pBR322 The present invention relates to detection, using a probe nucleotides, of nucleotide sequences derived from the origin of replication of the vector pBR322, present in therapy vectors genic.
Gene therapy is defined as the transfer of information genetic of therapeutic interest in a host cell or organism. The first protocol applied to humans was initiated in the United States in September 1990 on a genetically immunodeficient patient due d a mutation affecting the gene encoding 'Adenine Desaminase (ADA).
It was a question of correcting or replacing the defective gene whose dysfunction is at the origin of a genetic disease by a gene functional. The relative success of this first experiment encouraged the development of this technology which has since been extended to treatment of other genetic as well as acquired diseases (cancers, infectious diseases like AIDS ...) in order to deliver in situ therapeutic genes. Most strategies use vectors to to convey the therapeutic gene to its cell target. Many both viral and synthetic vectors have been developed during these the past few years and have been the subject of numerous publications accessible to The man of the trade (see for example Bobbins et al, 1988, Tibtech, 16, 34-40 and Rolland, 1998, Therapeutic Drug Carrier Systems, 15, 143-198).
As part of the monitoring of gene therapy in a patient, a which a drug in the form of a vector carrying a gene of interest therapy has been administered, it is important to monitor different parameters and in particular the following: the presence or (absence of the vector or even of its degradation products and its location.
The traceability of the vector indeed presents many benefits - clinical because the demonstration of the disappearance of the vector may cause doctor to adjust treatment and prescribe a new one administration of said vector,

2 - regulatory and ethical because it allows monitoring of the future drug vector vivo.
It is therefore advisable to have tests to detect the presence of the administered vector, or at least fragments thereof.
Currently, diagnostic tests are practical by certain laboratories associated with the place of treatment and by companies biotechnology service, according to protocols that are not standardized, are not specific, nor optimized, which do not meet the conditions of good laboratory practice and which lead to little results reproducible which do not allow their systematic use in the part of the follow-up of a treatment by gene therapy or more particularly in the case of controls of in vivo and ex vivo dissemination of vectors or in the case of preclinical and clinical studies.
To satisfy the need for a test not presenting the aforementioned drawbacks, the object of the present invention is to provide a test reliable, sensitive detection, applicable to a large number of vectors used in gene therapy protocols.
Plasmid pBR322 (GenBank Accession Number 27-4902-01), derived from the wild plasmid ColE1, is commonly used as starting material for the preparation of many vectors of genetical therapy. PBR322 is well characterized and has over thirty unique restriction sites, which makes it particularly advantageous in the design of new vectors [F. Bolivar et al, Gene 2, 95 (1977) and N. Watson, Gene, 70, 399 (1988) 7.
In particular, the pBR322 region comprising its origin from replication, served as the basis for the construction of numerous vectors of genetical therapy.
According to the invention, a detection tool and method are provided.
a nucleotide sequence derived from the origin of replication of said vector, applicable to any vector derived from pBR322.
The plasmid ColE1, of which derivative pBR322, being omnipresent in Man, it is also essential to be able to distinguish the modified form origin of replication, characteristic of the presence of a vector including the origin of replication of pBR322, the wild form of The origin of replication, to obtain a specific test. The specificity is a another advantage of the test proposed according to the invention.

WO 00/5380

3 PCT / FR00 / 00543 Thus, a first object of the invention is a fragment single-stranded nucleotide comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, in high stringency conditions, at the sequence defined by SEQ ID NO: 1 or a its complementary sequence. Preferably, said sequence comprises at minus twelve contiguous nucleotide motifs of a sequence chosen from SEQ ID NO: 1 and its complement. In the context of this invention, such a fragment preferably constitutes a probe.
The sequence SEQ ID NO: 1 described at the end of the description in the sequence list, corresponds to the complementary DNA sequence beginning at nucleotide 2140 and ending at nucleotide 3145 of the plasmid sequence available in databases (GenBank, www.ncbi.nlm.nih.gov/) sounds the access number J01749, and SEQ ID NO: 2 corresponds to that of the plasmid ColE1, for the same region (number J01566 in the same bank).
Before explaining in more detail (the invention, different terms used in the description and the claims are defined below - by “vector derived from pBR322”, is meant to designate the recombinant nucleic acid constructs of which at least the origin of replication is from pBR322. According to a particular case of the invention, a such vector will consist of a plasmid, it being further understood that said vector may further contain heterologous nucleic acid sequences a pBR322 and in particular nucleic sequences or acid constructs nuclei of therapeutic interest.
Advantageously, the gene of therapeutic interest codes for a Antisense RNA, a ribozyme, or even a polypeptide of interest. It can be from a eukaryotic organism, a prokaryote from a parasite or a virus.
It can be isolated by any conventional technique in the field of wax (by cloning, PCR, chemical synthesis ....). It can be of genomic type (comprising all or part of ('all of the introns), of DNA type complementary (cDNA, devoid of intron) or mixed type (minigene). Through elsewhere, the polypeptide for which it codes can be (i) intracellular, (ii) incorporated into the membrane of the host or (iii) secret cell. It can be a polypeptide as found in nature (native), a portion of it ci (truncate, of a mutant exhibiting in particular biological properties

4 improved or modified or a chimeric polypeptide from the fusion of sequences of various origins.
Among the polypeptides of interest which can be used, there may be mentioned more especially chemokines (MIP-1 a, MIP-1 b, RANTES, DC-CK1, MDC, MCP1 (monocyte chemoattraction protein), IP10 ....), cytokines (interferon a, b or g, interleukin (IL), in particular IL-2, IL-6, IL-10 or still IL-12, colony stimulating factor (GM-CSF, C-CSF, M-CSF) ...), cellular receptors (notably recognized by the HIV virus), ligands clotting factors (Factor VIII, Factor IX, thrombin, protein C), growth factors, factors proangiogenic (FGF for Fibroblast Growth Factor, VEGF for Vascular Endothelial Growth Factor, SH / HGF for scatter factor / Hepatocyte growth factor, TGF for transforming growth factor, TNF for necrotizing factor tumors, angiopoietin), enzymes (urease, repine, metalloproteinase, nitric oxide synthetase NOS, SOD, catalase, lecithin cholesterol acyl transferase LCAT ...), enzyme inhibitors (a1-antitrypsin, antithrombin III, viral protease inhibitor, PAI-1 for plasminogen activator inhibitor), major histocompatibility class I or II antigens or polypeptides acting on the expression of the corresponding genes, the antigens (or antigenic peptides) capable of generating a response immune, polypeptides capable of inhibiting wale infection, bacterial or parasitic or its development, the effect polypeptides antitumor (expression products of tumor suppressing genes, antigens associated with tumors, ...) positively acting polypeptides or negatively on apoptosis (Bax, Bcl2, BcIX ...), cytostatic agents (p21, p 16, Rb), the immunoglobulins in whole or in part (Fab, ScFv ...), toxins, immunotoxins, apolipoproteins (ApoAl, ApoAIV, ApoE ...), 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 for the target condition.
More specifically, the purpose of treating a dysfunction hereditary, we will use a functional copy of the defective gene, by example a gene coding for factor VIII or IX in the framework of Hemophilia A or B, dystrophin (or minidystrophin) within the framework of Duchenne and Becker's myopathies, insulin in the context of diabetes, CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator) in part of cystic fibrosis. With regard to inhibiting (initiation or progression of tumors or cancers, we will preferably implement a gene of interest coding for an antisense RNA, a ribozyme, a product

5 cytotoxic (thymidine kinase of herpes simplex virus 1 (TK-HSV-1), ricin, cholera toxin, diphtheria, product of yeast genes FCY1 and FUR1 encoding uracyl phosphoribosyl transferase and cytosine desaminase .....), an immunoglobulin, an inhibitor of cell division or transduction signals, a gene expression product tumor suppressor (p53, Rb, p73, DCC ....), a stimulator polypeptide of the immune system, an antigen associated with a tumor (MUC-1, BRCA-1, early or late papilloma virus antigens), possibly in combination with a cytokine gene. Finally, as part of a therapy anti-HIV, we can use a gene encoding a polypeptide immunoprotective, an antigenic epitope, an antibody (2F5; Buchacher and al., 1992, Vaccines 92, 191-195), the extracellular domain of the receptor CD4 (sCD4; Traunecker et al., 1988, Nature 331, 84-86) a immunoadhesin (e.g. CD4-IgG immunoglobulin hybrid; Capon et al., 1989, Nature 337, 525-531; Byrn et al., 1990, Nature 344, 667-670), an immunotoxin (e.g. fusion of the 2F5 antibody or Immunoadhesin CD4-2F5 to angiogenin; Kurachi et al., 1985, Biochemistry 24, 5494-5499), a trans-dominant variant (EP 0614980, W095 / 16780); a cytotoxic product such as one of those mentioned above above or an IFNa or (3.
According to the invention, said vectors or construction of acid nucleic acid usable in gene therapy, can be in their naked form (Wolff et al., 1990, Science 2, pages 1465-1468.), Associated with liposomes, cationic lipids, cationic polymers, peptides, polypeptides. Literature relating to vectors usable in therapy genique provides a large number of examples of such vectors (see par example Bobbins et al, 1988, Tibtech, 16, 34-40 and Rolland, 1998, Therapeutic Drug Carrier Systems, 15, 143-198).
- “nucleotide sequence” means a sequence single-stranded or double-stranded, linear or circular DNA;
- a "nucleotide fragment", and an "oligonucleotide" are two synonymous terms designating a chain of patterns

6 nucleotides characterized by the informational sequence of acids natural (or possibly modified) nucleic acids which are likely to hybridize, like natural nucleic acids, with a fragment complementary or substantially complementary nucleotide, in predetermined conditions; The sequence can contain patterns nucleotides with a structure different from that of nucleic acids natural; a nucleotide fragment (or oligonucleotide) contains usually at least 12 nucleotide units and can be obtained from from a natural and / or recombinant nucleic acid molecule genetic and / or by chemical synthesis;
- a nucleotide motif is derived from a monomer which can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is deoxy-2-ribose, in RNA sugar is ribose; as it 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; at as an example, the modification can take place, either at the level of the bases, with modified bases such as inosine, methyl-5-desoxycytidine, desoxyuridine, dimethylamino-5-desoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base capable hybridization, either at the sugar level, for example the replacement of at least one deoxyribose with a polyamide [PE Nielsen et al, Science, 254, 1497-1500 (1991)], again at the phosphate group level, by example its replacement with esters chosen in particular from diphosphates, alkyl- and aryl-phosphonates and phosphorothioates;
- by "informational sequence", we mean any sequence order of nucleotide-type patterns, the chemical and The order in a sense of reference constitutes 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 likely to associate 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 especially

7 more specific that it is performed at higher stringency; stringency depends in particular on the base composition of a duplex probe / target, as well as the degree of my pairing between two acids nucleic; stringency can also be a function of the parameters of hybridization reaction, such as concentration and type of species ionic present in the hybridization solution, the nature and concentration of denaturing agents and / or hybridization temperature; the stringency of the conditions under which a hybridization reaction is required to be carried out depends in particular on the probes used; all these data are well known and the appropriate conditions may be determined in each case by routine experiments; in general, depending on the length of the probes used, the conditions of strong stringency are as follows: the hybridization reaction is performed at a temperature between about 20 and 65 ° C, in particular between 35 and 65 ° C, in saline at a concentration of about 0.3 to 1M;
in particular, under the hybridization conditions according to the present invention, we choose a temperature of 37 ° C ~ 1 ° C, daps a solution saline PBS 3x (0.45 M NaCl; 0.15 M sodium phosphate);
- A "probe" is a nucleotide fragment comprising by example of 12 to 100 nucleotide units, in particular 12 to 35 units nucleotides, having specific hybridization under conditions determined to form an acid hybridization complex target nucleic acid comprising, in the present case, a sequence nucleotide derived from the origin of replication of the vector pBR322; a probe can in particular be used for diagnostic purposes, under the in particular form 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 present in any suitable form such as tube, cone, well, plate microtitration, sheet, soluble polymer; it is made up of a material natural, synthetic, modifies chemically or not and is, depending on the selected technique, chosen from polystyrenes, styrene copolymers-butadiene, styrene-butadiene copolymers mixed with polystyrenes, polypropylenes, polycarbonates, copolymers polystyrene-acrylonitrile, styrene-methylmethacrylate copolymers of

8 methyl, among nylon and natural synthetic fibers, among polysaccharides and cellulose derivatives;
- a "detection probe" can be marked with a marker chosen for example from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic substrate, fluorigenic or luminescent (in particular a peroxidase or a phosphatase alkaline), chromophoric chemical compounds, chromogenic, fluorigenic or luminescent, base analogs nucleotides, and ligands such as biotin;
- a "primer" is a probe comprising for example 12 has 100 nucleotide motifs and having a specificity of hybridization in conditions determined for the initiation of polymerization enzymatic, for example in an amplification technique.
The probes according to the invention are used for the purpose of diagnosis, in the search for the presence or (absence of a target nucleotide sequence in a sample, according to all known hybridization techniques and in particular deposition techniques point on filter, say "DOT-BLOT" (MANIATIS et al, Molecular Cloning, Cold Spring Harbor, 1982), DNA transfer techniques say "SOUTHERN BLOT" [SOUTHERN. EM, J. Mol. Biol., 98, 503 (1975)], or the so-called "sandwich" techniques [DUNN AR, HASSEL JA, Cell, 12, 23 (1977)]; the sandwich technique is advantageously used, with at least one capture probe and / or at least one capture probe detection; in the case where both types of probes are used, At least one of said probes (generally the detection probe) is able to hybridize with a target region that is specific for the sequence sought, it being understood that the capture probe and the detection must have at least nucleotide sequences partially different so that said probes are capable to hybridize with two different regions of the target nucleic acid.
The main stages of the so-called sandwich technique first consist in immobilizing a capture probe on a support, in particular by passive or covalent adsorption, to bring the or the probe (s) immobilized with a sample, possibly pretreated, likely to contain at least one nucleotide sequence to be detected, so-called target sequence, under conditions allowing hybridization of the or

9 probes with a target sequence complementary to said one or more probe (s), and detecting the hybridization complex formed between them.
The detection of the complex is carried out by contacting this one with a detection probe, marked by a labeling agent, puffs direct or indirect detection, in particular by revealing the agent marker pen. The detection probe can of course be present as soon as first step of the sandwich protocol.
According to a particular case of the invention, the detection probe is not marked. In this case the detection of the probe / acid complex target nucleic acid can be achieved by the use of detection means specific nucleic acids are double stranded. For this purpose and as an example, it is possible to use antibodies capable of recognize double strand structures of nucleic acids (WO-A-97/32602).
The different objects of the invention and their variants preferential are explained below.
As mentioned previously, a fragment of the invention includes at least twelve nucleotide motifs capable of hybridizing to a region of SEQID NO: 1 or its complement, under conditions high stringency defined above.
Excluded from the invention are fragments whose sequence is chosen from the sequence of plasmid pBR322 available in GenBank under the access number J01749 and SEQ ID NO: 44 to 47.
Nucleotide sequences derived from the origin of replication of pBR322 being well preserved, a preferred fragment of the invention includes, or consists of, at least twelve contiguous nucleotide units belonging to SEQ ID NO: 1, and more advantageously still belonging has a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 43 and the complementary to SEQ ID NO: 3 to SEQ ID NO: 43.
A second object of the invention is a nucleotide probe for capture and / or detection of a gene therapy vector or degradation products of such a vector, and more particularly of a nucleotide sequence derived from the origin of replication of pBR322, which includes or consists of a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide motifs likely to hybridize, under conditions of high stringency, has 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 SEO ID NO: 3 to SEQ ID NO: 43, and their supplements. A probe for the specific detection of a so-called nucleotide sequence is 5 advantageously chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complementary to SEQ ID NO: 23 to SEQ ID NO: 43.
For the implementation of the so-called detection technique “Sandwich” in particular, we defined according to the invention of probes capture which preferably includes or consists of a sequence

10 chosen from SEQ ID NO: 3 to SEQ ID NO: 22 and the additional ones SEQ ID NO: 3 to SEQ ID NO: 22, and detection probes which preferably include or consist of a sequence chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complements of SEQ ID NO: 23 a SEQ ID NO: 43.
Another object of the invention is a primer for amplification enzymatic, 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 includes or consists of a fragment single-stranded nucleotide comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, in high stringency conditions, at 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 complements. In particular it includes or consists of a sequence chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complementary to SEQ ID NO: 23 to SEQ ID NO: 43.
The invention also relates to a reagent for detecting and / or identify and / or quantify 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 previously, and possibly at least one primer of the invention.
Another object of the invention is a method for detecting and / or identify and / or quantify 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

11 biological, likely to contain at least one said sequence nucleotide. It includes the stages consisting (a) putting said sample in contact with at least a probe of the invention, under conditions allowing the formation of a hybridization complex, and (b) detect, by any appropriate means, the formation of a hybridization complex between said probe and said nucleotide sequence.
The detection method is advantageously a technique "Sandwich". 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 detection probe of the invention. II
favorably includes a preliminary step of amplifying said nucleotide sequence, which is preferably carried out by contacting of the sample with a primer described above.
Step (b) can be carried out using an antibody, possibly coupled with a marking agent, directed against said company hybridization.
The invention also relates to the use of a probe and / or a primer previously described, to determine the presence or ('absence a nucleotide sequence derived from the origin of replication of pBR322 or a vector and / or a vector fragment comprising such a sequence nucleotide in a biological sample.
An appropriate biological sample is chosen in particular from a fluid type sample (such as blood, urine), tissue or fragment of tissue, mucositis, organ or organ fragment, or culture supernatant obtained using one of the abovementioned samples. Preferably, such a biological sample comes from a patient previously treated with a gene therapy protocol using a vector as defined according to the present invention. According to a particular case, such a sample consists of a blood sample but it is preferred in the context of present invention take samples of pulmonary origin or muscular. More specifically, such a sample is taken from the site administration of the vector during the implementation of the genetical therapy.

12 But ('use according to' the invention may also relate to 'study the dissemination of a vector derived from pBR322, in particular daps The patient's body, in the environment or in the products products from a patient treated by gene therapy. In particular, when such a patient wishes to donate his blood or one of his organs, ('use according to' the invention will make it possible to determine in a way reliable whether or not the biological material donated by the patient contains a pBR322 derivative vector. The use according to the invention is based on this in an ever-increasing need for security when donating equipment organic.
It is also possible to consider applying the method of detection of the invention in the food industry or cosmetic. In fact, these fields are calling more and more often for the development of their products for the use of organisms genetically modified (GMO), i.e. transformed using a recombinant vector. The method of the invention therefore provides a reliable means and effective in determining whether a food or cosmetic product has been developed using a GMO.
The invention is illustrated by the following example of detection of a vector derived from pBR322 by the “sandwich” technique, after amplification.
Example: Transfection of cells with the plasmid pCH 104 293 cells are transfected with the plasmid pCH 104 (Hall et al., J. of Molecular and Applied Genetics, 1983, 2, 101-109) including The origin of replication derived from pBR322 using the Calcium Phosphate kit GIBCO-BRL Transfection System following the manufacturer's instructions.
~ DNA extraction Plamid DNA is extracted from cells by the protocol HIRT, where the cells are lysed with the following buffer 24 L. ~ I from SDS 10 8 µl EDTA 0.5 M
8 q, l proteinase K at 10 mg / ml 160 ~ I of TE pH8 We then carry out an extraction with phenol / chloroform puffs 100% ethanol precipitation. After centrifugation, the DNA pellet plasmid is taken up in an RB buffer.

13 ~ PCR amplification The primers used for the amplification reaction include the following sequences SEQ ID NO: 26 SEQ ID NO: 38 The amplification reaction is carried out under the conditions detailed below. The total volume of the reaction is 100 μl. II
includes a 50 mM Tris-HCl pH 8.5 buffer, containing 4 mM MgCl2, 100 ~ g / ml of BSA (bovine serum albumin), 1 p, M of each primer, 200 µM of each dNTP and 1 U of Taq polymerase. The reaction medium is then subjected to a first series of 5 puff cycles to a second series of 30 amplification cycles. In the first series, DNA is first denature by incubating the samples at 94 ° C for 10 seconds, puffs at 55 ° C for 10 seconds, so that the primers can pair with the DNA template, and finally at 72 ° C for 30 seconds to allow extension of the primers. In the second series, the cycles are as follows: 94 ° C for 10 seconds, puffs 60 ° C
for 10 seconds and 72 ° C for 30 seconds.
~ Detection on microplate Capture and detection probes are chosen in a way 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 has a peroxidase enzyme group which will allow evidence the formation of hybridization complexes. This marking is carried out according to the procedure described in PCR protocols: A guide to methods and application; Academic Press (1990), 15, p4513-4534. The activity enzymatic 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, 16, 4077-4095, 1988) or by covalent coupling (Rasmussen, et al, 1991, Analytical Biochemistry, 198, 138-142).
The product of the amplification reaction is denatured in a NaOH / EDTA solution, puffs introduced using a buffer solution hybridization in the microplate wells at the same time as the probe of detection.

14 After an incubation phase (1 hour at 37 ° C with shaking) and a washing phase (100 mM Tris, 3M NaCI, 1% Tween 20, pH 7.4), Peroxidase activity linked to the detection probe is detected by colorimetry. This detection is carried out by means of a solution of trisodium citrate and orthophenylenediamine (OPD). After incubation of 30 minutes in the dark, a 4N H2S04 solution is added to stop fa reaction. The optical density is then determined at 492 nm.

LIST OF SEQUENCES
<110> TRANSGENE
<120> Nucleotide fragment, probe, primer, reagent and method for detecting a derived nucleotide sequence origin of replication of pBR322 <130> vector detection pBR322 <140>
<141>
<150> FR9902968 <151> 1999-03-05 <160> 47 <170> PatentIn Ver. 2.1 <210> 1 <211> 995 <212> DNA
<213> pBR322 Plasmid <400> 1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 60 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 120 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 180 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 240 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 300 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 360 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 420 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 480 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 540 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 600 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctcacat 660 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 720 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 780 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 840 gtgcactcta cgtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta 900 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc 960 tgacgggctt gtctgctccc ggcatccgct tacag 995 <210> 2 <211> 997 <212> DNA
<213> ColEl Plasmid <400> 2 gtagaaaaga ttaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 60 caaacaaaaa aaccaccgct accaacggtg gtttgtttgc cggatcaaga gctaccaact 120 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 180 tagccgtagt cgggccacta cttcaagaac tctgtagcac cgtttgtgcc atcatcgctc 240 5 5 tgctaatccg gttaccagtg gctgctgcca gtggcgttaa ggcgtgcctt accgggttgg 300 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 360 cacagcccag cttggagcga acgacctaca ccgaactgag ataccaacag cgtgagctat 420 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 480 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtag ctttatagtc 540 ctgtcgggtt tcgccacctc tgacttgagc gtctattttt gtgatgctcg tcaggggggc 600 ggagcctatg gaaaaacgcc tgctacgtgg ccttcttcct gttcctggtc ttttgctcac 660 atgttctttc cggccttatc ccctgattct gtggataact gtgttaccgt ttttgtgtga 720 gtcagtaccg ctcgccgcag tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg 780 gaaaagcgcc tggacgtgca ttttctcctt acgcatctgt gcggcatttc acacccggca 840 tggcgtactt ttcatacaat ccgcactgat gccgcatggt taagccagta tacactccgc 900 tatcgctacg tgactgggtc agggctgcgc cccgacaccc gctaaaacct gctgacgcgc 960 cctgacgggc ttgtcagctc ccggcatccg ctcacag 9g7 <210> 3 <211> 80 <212> DNA
<213> pBR322 Plasmid <400> 3 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 60 caaaaaaacc accgctacca gp <210> 4 <211> 105 <212> DNA
<213> pBR322 Plasmid <400> 4 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 60 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagt 105 <210> 5 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 5 acttcaagaa ctctgtagca ccg 23 <210> 6 <211> 15 <212> DNA
<213> pBR322 Plasmid <400> 6 tcgctctgct aatcc 15 <210> 7 <211> 26 <212> DNA
<213> pBR322 Plasmid <400> 7 gttaccagtg gctgctgcca gtggcg 26 <210> 8 <211> 118 5 5 <212> DNA
<213> pBR322 Plasmid <400> 8 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 60 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagatacc 118 <210> 9 <211> 123 <212> DNA
<213> pBR322 Plasmid <400> 9 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 60 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 120 gta 123 <210> 10 <211> 54 <212> DNA
<213> pBR322 Plasmid <400> 10 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcacttgag cgtc 54 <210> 11 <211> 46 <212> DNA
<213> pBR322 Plasmid <400> 11 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgcc 46 <210> 12 <211> 22 <212> DNA
<213> pBR322 Plasmid <400> 12 cttttgctca catgttcttt cc 22 <210> 13 <211> 24 <212> DNA
<213> pBR322 Plasmid <400> 13 ttatcccctg attctgtgga taac 24 <210> 14 <211> 15 <212> DNA
<213> pBR322 Plasmid <400> 14 taccgctcgc cgcag 15 <210> 15 <211> 14 <212> DNA
<213> pBR322 Plasmid <400> 15 cgaacgaccg agcg 14 <210> 16 <211> 27 <212> DNA

<213> pBR322 Plasmid <400> 16 agcgagtcag tgagcgagga agcggaa 27 <210> 17 <211> 25 <212> DNA

<213> pBR322 Plasmid <400> 17 attttctcct tacgcatctg tgcgg 25 <210> is <211> 12 <212> DNA

<213> pBR322 Plasmid <400> is ctgatgccgc at 12 <210> 19 <211> 43 <212> DNA

<213> pBR322 Plasmid <400> 19 gttaagccag tatacactcc gctatcgcta cgtgactggg tca 43 <210> 20 <211> 20 <212> DNA

<213> pBR322 Plasmid <400> 20 ggctgcgccc cgacacccgc 20 <210> zl <211> 14 <212> DNA
<213> pBR322 Plasmid <400> 21 acgggcttgt ctgc <210> 22 <211> 16 5 5 <212> DNA
<213> pBR322 Plasmid <400> 22 gctcccggca tccgct 16 <210> 23 <211> 23 5 <212> DNA
<213> pBR322 Plasmid <400> 23 gtagaaaaga tcaaaggatcttc 23 <210> 24 <211> 23 <212> DNA

<213> pBR322 Plasmid <400> 24 caccgctacc agcggtggtttgt 23 <210> 25 <211> 31 <212> DNA

<213> pBR322 Plasmid <400> 25 2 gtagccgtag ttaggccaccacttcaagaa c 31 <210> 26 <211> 32 <212> DNA

<213> pBR322 Plasmid <400> 26 ctgtagcacc gcctacatacgtcgctctgc to 32 <210> z ~

<211> 23 <212> DNA

<213> pBR322 Plasmid <400> 2 ~

tctgctaatc ctgttaccagtgg 23 <210> 28 <211> 33 4 <212> DNA

<213> pBR322 Plasmid <400> 28 tgccagtggc gataagtcgtgtcttaccgg gtt 33 <210> 29 <211> 23 <212> DNA

<213> pBR322 Plasmid <400> 29 actgagatac ctacagcgtgagc 23 <210> 30 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 30 aacgcctggt atctttatag tcc 23 <210> 31 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 31 acttgagcgt cgatttttgt gat 23 <210> 32 <211> 61 <212> DNA
<213> pBR322 Plasmid <400> 32 ggaaaaacgc agcaacgcgg cctttttacg gttcctggcc ttttgctggt cttttgctca 60 c 61 <210> 33 <211> 26 <212> DNA
<213> pBR322 Plasmid <400> 33 atgttctttc ctgcgttatc ccctga 26 <210> 34 <211> 4a <212> DNA
<213> pBR322 Plasmid <400> 34 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgcc 4g <210> 35 <211> 23 <212> DNA
4 5 <213> pBR322 Plasmid <400> 35 gctcgccgca gccgaacgac cga 23 <210> 36 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 36 acgaccgagc gcagcgagtc agt 23 <210> 37 <211> 38 <212> DNA
<213> pBR322 Plasmid <400> 3 ~
aggaagcgga agagcgcctg atgcggtatt ttctcctt 38 <210> 38 <211> 63 <212> DNA
<213> pBR322 Plasmid <400> 38 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 60 gca 63 <210> 39 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 39 tgatgccgca tagttaagcc agt 23 <210> 40 <211> 23 <212> DNA
<213> pBR322 Plasmid <400> 40 tgactgggtc atggctgcgc ccc 23 <210> 41 <211> 44 <212> DNA
<213> pBR322 Plasmid <400> 41 4 ~ ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtc 44 <210> 42 <211> 20 <212> DNA
4 5 <213> pBR322 Plasmid <400> 42 ggcttgtctg ctcccggcat 5 ~ <210> 43 <211> 16 <212> DNA
<213> pBR322 Plasmid 55 <400> 43 cggcatccgc ttacag 16 <210> 44 <211> 21 <212> DNA
<213> pBR322 Plasmid <400> 44 acgatagtta ccggataagg cgcagcggtc gggctgaacg g 21 <210> 45 <211> 180 <212> DNA
<213> Artificial sequence <400> 45 cctttgagtg agctgata lg <210> 46 <211> 24 <212> DNA
<213> Artificial sequence <400> 46 tattttctcc ttacgcatct gtgc 24 <210> 47 <211> 21 <212> DNA
<213> Artificial sequence <400> 47 gatgccgcat agttaagcca g 21

Claims (20)

1. 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 its complement, excluding fragments whose sequence is chosen from the sequence of the plasmid pBR322 available in GenBank under accession number J01749 and SEQ ID NOS: 44 to 47. 2. Fragment according to claim 1, characterized in that that said sequence comprises at least twelve nucleotide motifs contiguous with a sequence chosen from SEQ ID NO: 1 and its additoinal. 3. Fragment according to claim 2, characterized in that that said sequence comprises at least twelve nucleotide motifs contiguous with a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 43 and the complements of SEQ ID NO:3 to SEQ ID NO:43. 4. Fragment according to claim 2, characterized in that that said sequence consists of a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 43 and the complements of SEQ ID NO:3 to SEQ ID NO:43. 5. Probe for the capture and/or detection of a nucleotide sequence derived from the origin of replication of the vector pBR322, characterized in that it comprises or consists of a single-stranded nucleotide fragment comprising a sequence of at at least twelve contiguous nucleotide motifs capable of hybridizing, under high stringency conditions, to SEQ ID NO:1 or its complementary, and preferably, to 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 complements. 6. Probe for vector capture and/or detection and/or a vector fragment comprising a nucleic sequence derived from the origin of replication of pBR322, characterized in that it comprises or consists of a single-stranded nucleotide fragment comprising a sequence of at least twelve nucleotide units contiguous areas susceptible to hybridization, under conditions of high stringency, has SEQ ID NO: 1 or its complement, and preferably, to a fragment comprising or consisting of a selected sequence from SEQ ID NO: 1 and SEQ ID NO: 3 to SEQ ID NO: 43, and their complementary. 7. Probe according to claims 5 and 6, characterized in what it is a capture probe and what it includes or consists of a sequence chosen from SEQ ID NO: 3 to SEQ ID NO: 22 and the complements of SEQ ID NO:3 to SEQ ID NO:22. 8. Probe according to claim 7, characterized in that that it is immobilized on a solid support. 9. Probe for sequence-specific detection nucleotide derived from the origin of replication of pBR322 according to the claim 5 or of a vector and/or a vector fragment according to 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 complementary to SEQ ID NO:23 to SEQ ID NO:43. 10. Probe according to claim 9, characterized in that that it is labeled with a labeling agent, in particular chosen from radioactive isotopes, enzymes, especially enzymes capable of acting on a chromogenic, fluorogenic or luminescent such as peroxidase or alkaline phosphatase, chromophoric chemical compounds, chromogenic compounds, fluorogenic or luminescent, analogues of nucleotide bases and ligands such as biotin. 11. Primer for enzymatic amplification of at least a nucleotide sequence derived from the origin of replication of pBR322, characterized in that it comprises or consists of a single-stranded nucleotide fragment comprising a sequence of at least twelve contiguous nucleotide motifs capable of hybridizing, under high stringency conditions, to SEQ ID NO:1 or its complementary, and preferably, to 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 complements. 12. Primer for specific enzyme amplification of at least one nucleotide sequence derived from the origin of replication of pBR322 according to claim 11, characterized in that that it comprises or consists of a sequence chosen from SEQ ID NO: 23 to SEQ ID NO: 43 and the complements of SEQ ID NO:23 to SEQ ID NO:43 13. Reagent to detect and/or identify and/or quantify 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 at least one capture probe according to claim 6 or 7 and at least a detection probe according to claim 9 or 10. 14. Reagent according to claim 13, characterized in that that it further comprises a primer according to claim 11 or 12. 15. 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 containing a said nucleotide sequence, in a biological sample, capable of containing at least one said nucleotide sequence, or a said vector and/or a said vector fragment characterized in that it includes the steps consisting of:

(a) bringing said sample into contact with at least one probe according to any one of claims 5 to 10, under conditions permitting the formation of a hybridization complex, and (b) to detect, by any appropriate means, the formation of a hybridization complex between said probe and said nucleotide sequence. 16. Method according to claim 15, characterized in that that said sample is brought into contact with at least a first probe according to claim 6 or 7, and at least one second probe according to claim 9 or 10. 17. Method according to claim 15 or 16, characterized in that it comprises a preliminary step of amplifying said nucleotide sequence. 18. Method according to claim 17, characterized in that that said sample is brought into contact with at least one primer according to claim 11 or 12. 19. Method according to claim 15, characterized in that that step (b) is carried out using an antibody, optionally coupled to a marker agent, directed against said hybridization complex. 20. Use of a probe according to any of the claims 5 to 10 and/or a primer according to claim 11 or 12, to determine the presence or absence of a sequence nucleotide derived from the pBR322 origin of replication of a vector and/or a vector fragment comprising said sequence nucleotide, in a biological sample.