FR2555606A1 - Non-radioactive probe and process for the characterisation of a given nucleotide sequence and means for the production of this non-radioactive probe - Google Patents

Abstract

The invention relates to a non-radioactive probe for the detection, by hybridisation, of a given nucleotide sequence and comprising, by way of essential constituent, a nucleic acid itself containing an insert comprising a sequence complementary to the said given nucleotide sequence. This probe is characterised in that at least a portion of at least one of the constituent nucleotides of its nucleic acid is replaced by modified bases recognisable by polymerases and containing a group comprising at most two carbon atoms and, preferably, at least one halogen atom.

Description

Non-radioactive probe and method for characterizing a determined nucleotide sequence and means for producing this non-radioactive probe.

 The invention relates to a new specific non-radioactive labeled probe and to a method for the detection by hybridization of the presence and, optionally, of the characterization of a determined nucleotide sequence within a composition based on nucleic acids. (DNAs, cDNAs, RNAs), this labeled non-radioactive probe itself containing a nucleotide sequence complementary to the previous one and capable of hybridizing under the appropriate conditions with said nucleotide sequence determined to be detected. The invention also relates to a method and to means for the manufacture of such a non-radioactive probe. The invention finally relates to applications in many fields of this new non-radioactive probe, these applications generally having all in common the isolation from a biological sample of its nucleic acid content and the detection of the presence or not, within these nucleic acids (DNA, RNA or both at the same time) of the nucleotide sequence determined by hybridization with the complementary sequence contained in the probe according to the invention.

 The specific DNA probes, still the most commonly used to date for identifying DNA-DNA or DNA-RNA hybridization in hybridization experiments carried out either in vitro or in situ, are labeled with isotopes and involve autoradiographic detection.

 The main disadvantages of autoradiographic detection of hybridization are the dangers run by the experimenter when handling radioactive products, the low resolving power, the imprecision of the localization, the length of the exposure time of the films ( from several hours to several weeks) and the instability of the probes due to radiolytic decomposition.

 To remedy this difficulty, it has already been proposed to replace radioactive labeling by other labeling methods. In particular, the technique described for the first time in French patent No. 78 10975 filed on April 13, 1978 will be recalled.

 The method described in the aforementioned patent for the detection of the possible presence or characterization of a determined sequence or fragment of nucleic acid, in particular of a gene, or even of the entire nucleic acid within a sample of complex nucleic acids, by bringing the sample into contact, if necessary after prior denaturation of the nucleic acid studied, with a probe comprising a complementary nucleic acid, capable of hybridizing with the nucleic acid sequence or the nucleic acid sought, is characterized in that the probe used is a probe modified chemically by coupling or with a view to its coupling with an enzyme, preferably after the hybridization reaction, the possible presence of the sequence nucleic acid or nucleic acid sought after then being revealed by the action of the hybridization product formed from the probe and the sequence or nucleic acid sought, on a substrate of the enzyme.

 Advantageously, the enzyme is chosen according to its capacity to act on a chromogenic substrate, which makes it possible to assay, by optical analysis or the like, the rate of transformation of the substrate, a rate which is then correlable to the presence or not of the sequence d nucleic acid or nucleic acid sought in the initial sample.

In a preferred embodiment of the invention of the patent, the probe is modified by a group
chemical capable of forming a stable complex with
the enzyme or a molecule itself stably linked to the enzyme. Advantageously, the aforementioned chemical group and the aforementioned molecule are respectively constituted by biotin.e and avidin or vice versa, the enzyme itself being constituted for example by beta-salasidase, glucose oxidase, alkaline phosphatase , peroxidase.

 The detection or "revelation" of the hybridized probe can use means distinct from the enzyme, for example immunofluorescence techniques.

The coupling caused between the hybridized probe and the enzyme or other separate development means used in the process recalled above can be carried out either directly (for example by means of the biotin-avidin reaction or of a reaction antigen-antibody), either indirectly, for example in a reaction sequence comprising by first contacting the hybridized probe carrying haptenic groups with antihapten antibodies, then a second contacting the hybridized probe which then retains antibodies anti-hapten, with immunoglobulins or other affine molecules directed against immunoglobulins, themselves labeled according to one of the non-radioactive techniques which has been recalled or by equivalent techniques. There is no need to insist on the conditions under which these techniques can be implemented. They are well known. If necessary, we
will also refer to European patent application No. 0063869-A3 in which they were recalled.

It should also be noted, however, that this European patent application, among other features, is expanding. also the field of possibilities for labeling non-radioactive nucleic probes and describes nucleic acid probes in which certain bases carry substituents which, according to the teachings of this patent application, must contain at least three carbon atoms, and preferably even at least five atoms of. carbon, these groups being chosen from those which can be detected by a molecule, in particular a polypeptide demonstrating a great affinity for these groups, and this when the probe forms a duplex in double helix with a ribonucleic or deoxyribonucleic sequence complementary to a fragment of nucleotides contained in the probe,
The techniques which have just been recalled and which both use non-radioactive probes or, for the convenience of the language hereinafter "cold probes" (as opposed to "hot probes" which are probes marked with isotopes radioactive), have significant advantages over "hot probes", if only at the level of the greatest safety of the experimenters who use them. But there is still still a need to perfect them to make them both more effective and more sensitive.

In addition, it should be noted that the character which is both complex and costly to carry out the actual labeling of the probes in question firstly needs to synthesize modified nucleotides under the conditions which have been described in the patent, then incorporation. in vitro of these modified nucleotides, for example biotinyl-nucleotides, in the double-stranded chains of the nucleic acids to be marked by a technique carrying out openings and localized repairs of one of the chairs by means of a polymerase
(technique called "nick-translation".

It was also proposed in the request for
European patent to polymerize the modified nucleotide
previously obtained, for example biotinyl-dUTP, in
presence of a matrix ("template") including an ex
poly-A terminal end, in the presence of an RNAligase.

 The invention therefore aims to provide cold probes which meet the following objectives - the probes must hybridize easily to nucleic acids, - the modification of some of their nucleotide bases must be minimal, so that these probes remain capable of hybridize to strands of complementary nucleic acids, - the sensitivity of the method must be at least equal to that obtained with radioactive probes.

 Another object of the invention is to provide manufacturing means and techniques allowing these probes or more generally labeled DNAs to be obtained quickly and at relatively low cost.

In general, the cold probe according to the invention for the detection of a determined sequence of nucleotides by hybridization, and which itself comprises an insert comprising a sequence complementary to said nucleotide sequence, is characterized in that at least part of the bases of at least one of the types of nucleotides constituting its nucleic acid is replaced by modified bases recognizable by the polymerases,
and comprising a group comprising at most two atoms
of carbon. Preferably, this group comprises at least one halogen or is constituted by a halogen.

In general, we can implement
any modified base meeting the conditions
yield and which do not interfere with the WATSON-CRICK pairing capacity of the nucleic acid thus modified with a natural nucleic acid complementary or containing similar modified bases.

 Preferably, the modified bases are derived from natural nucleotide bases, the modification of which resides in a substitution with the aforementioned group, in position 5, when it is a pyrimidine base and in position 7 or in position 8, or in both positions at the same time, of a deazapurine or purine base.

 Preferred probes are those in which the modified bases are 5-halo-uridine or 5-halo-deoxy-uridine groups, and more particularly still in which these modified bases are 5-bromo-uridine or 5-group bromo-deoxy-uridine.

Other preferred groups capable of being used in the context of this invention involve corresponding derivatives substituted by iodine or fluorine, or also by CF3 groups.

 The cold probes according to the invention, which can, after hybridization, be subsequently detected by means of antibodies, preferably monoclonal antibodies directed selectively against the modified bases, have the clear advantage that they may contain a level of substitute of l 'one of their natural bases by corresponding modified bases, which could not be achieved to date in practice, even when the incorporations of these bases modified in double-stranded DNA by in vitro techniques, for example similar to those envisaged in European patent application No. 0063879 are used, and this in no way affects their capacity for hybridization (in particular appreciated by lowering the temperature Tm) with natural complementary nucleotide sequences.

 The selectivity in the antigen-antibody reaction, which involves each individually modified base, which may possibly in some cases - but certainly not all - be more difficult to achieve than in the case of probes according to the prior techniques, is then more than largely compensated for by the higher, even total, rate of substitutions of modified bases to the natural bases which can be carried out, without the slightest harm being done to the hybridization capacity of the probes formed with complementary DNAs.

dans laquelle
- chacune des lettres B, B', B"... représente un groupe purine, 7-déazapurine ou pyrimidine, chacun de ces groupes B, B', B"... étant lié de façon covalente au groupe sucre correspondant en la position C de celuici, soit au niveau de sa propre position N9, pour les groupes purine ou 7-déazapurine, soit au niveau de sa propre position N1 pour les groupes pyrimidines,
- A représente un groupe de modifications de B, B', B" , ....., selon le cas, consistant en un groupe de substitution comprenant au plus deux atomes de carbone et, de préférence, étant au surplus halogéné, ce groupe
A étant directement lié à lthétérocyle de la base azotée, soit en position 5, lorsque B, B', B" .... sont des groupes pyrimidine, soit en position 7, ou en position 8, ou dans les deux positions à la-fois, lorsque B, B',
B" ... sont des groupes déazapurine ou purine,
- z représente -H ou -OH et
- m et n représentent respectivement des nombres entiers de O à 100.000,
- p est un nombre entier de 1 à 75.000, étant entendu que la somme m + n + p est au moins égale à 2, de préférence au moins égale à 100.A class of probes in accordance with the invention, containing an insert as defined above, can also be defined by the structure

in which
each of the letters B, B ', B "... represents a purine, 7-deazapurine or pyrimidine group, each of these groups B, B', B" ... being covalently linked to the corresponding sugar group by position C of this, either at its own position N9, for the purine or 7-deazapurine groups, or at its own position N1 for the pyrimidine groups,
- A represents a group of modifications of B, B ', B ", ....., as the case may be, consisting of a substitution group comprising at most two carbon atoms and, preferably, being additionally halogenated, this group
A being directly linked to the heterocyle of the nitrogenous base, either in position 5, when B, B ', B ".... are pyrimidine groups, either in position 7, or in position 8, or in the two positions at the -times, when B, B ',
B "... are deazapurine or purine groups,
- z represents -H or -OH and
- m and n respectively represent whole numbers from O to 100,000,
- p is an integer from 1 to 75,000, it being understood that the sum m + n + p is at least equal to 2, preferably at least equal to 100.

 In the above, the term insert "does not necessarily mean that it is a heterologous nucleic acid sequence, that is to say of foreign origin, vis other components of the cold probe. The insert and other components can be of the same origin or be derived from a single nucleic acid, for example when the probe consists of an entire fragment which has been cloned into a appropriate vector and then excised therefrom, for example via a restriction enzyme to which no cleavage site corresponded in the DNA fragment in question, however this will not be the case in numerous cases, in particular each time that the probe will in fact consist of a recombinant plasmid or cosmid, or a recombinant phage DNA containing said insert, especially and obviously when the latter is found to be of eukaryotic origin.

It will also be noted that the modifications of at least one of the bases of the probe, under the conditions which have been specified, may not affect the whole of the probe. They can relate either to the insert itself, or to the other parts of the probe. This may be the case, each time the probe has been formed by genetic recombination in vitro, between fragments comprising modified bases and distinct fragments, in which the corresponding bases have not been modified. invention may also be concentrated on one end or on the two opposite ends of the probe, in particular when the latter (or these) will include (or will include) one or two double-strand (s), such as poly-A-poly-U attached to this (or these) end (s) via a
RNA ligase or an appropriate terminal transferase, the bases of either the poly-A strand or the oly-U strand, which can then be modified as indicated above
Among the preferred cold probes of the invention are those in which the modified bases used have been chosen from those which are biologically incorporated into the genomic DNA of a microorganism.

 In this regard, the invention takes advantage of the observations already made by certain authors and according to which deoxybromo-uridine- (BrdUd) can stincorpore in the DNA of eukaryotic cells in place of thymidine Q'att. , PNAS U.S., 70, 3395 (1979j.

On the other hand, it has been described that halogenated bases (5-bromo ~ uracil) for example could be incorporated into the DNA of bacteria in place of thymine (ZAMENHOF and GRIBOFF, Nature 1954) and that the yield of incorporation was improved using an dlE strain. thymine-dependent coli (HACKETT et al ,, Biochem. Biophys.

Acta. 123 (1966) 356-363). The preparation of antibodies recognizing the brominated bases has also been described (SAWICKI et al. Science, 174, 70 (1971). Finally, some authors have used these antibodies to locate the replication of DNA in the chromosomes having incorporated
BrUd: GRATZNER et al. Exp. Cell. Res. 95, 88 (1975) or even monoclonal antibodies (HG GRATZNER, Science, vol. 218, 1982.

But in these techniques, the micro strains
organisms obtained, having incorporated BrdUd have not
usually not shown to be stable, which for apps
cations envisaged, was also not necessary.

 The invention therefore also relates to a method of in vivo manufacturing of probes of the genus defined above in microorganisms, in particular bacteria, and more particularly E. coli.

 The method according to the invention for manufacturing the probe according to the invention, when the latter is derived from a recombinant DNA or from a vector DNA capable of transforming a microorganism, such as plasmid, cosmid or phage DNA and containing the aforementioned insert, is characterized by setting and maintaining in culture the microorganism previously transformed by this recombinant DNA with a culture medium containing the modified base replacing the corresponding natural base, the said modified base s' therein, either in the free state or in the combined state, in a nucleoside or nucleotide corresponding to said base, and by the subsequent recovery of DNA-recombinant or DNA-vector.

 For the preliminary transformation of the strains with the recombinant DNA or vector DNA, containing the above-mentioned insert and which, after the implementation of the method according to the invention, will provide the labeled probe, preferably strains better suited for use are used. particular requirements of the invention. In particular, these microorganisms are preferably stabilized to be cultivable over several generations, in the presence of the modified base and to be able to allow the substitution in vivo of at least one of the bases contained inside nucleic acids. , in particular the aforementioned DNA-recombinants and DNA-vectors previously introduced into the microorganism by genetic engineering techniques, by modified bases c3rresFndantes as specified above.

 Preferably, the microorganism initially used is a mutated bacterium, preferably an E. coli mutant.

 In general - the strain must be able to accommodate, for example by transformation using plasmids (but all other vectors, viruses in particular, must also be able to be used) or foreign DNA; it must therefore essentially be "restriction -" - the strain must be able to incorporate a high level of bases analogous to normal bases, and therefore the anabolism of normal bases must be reduced or eliminated, and the catabolism of nucleotides (normal or abnormal, or the like) must be greatly reduced or eliminated, by biochemical or genetic means, - the strain must be stable and the vectors introduced into the strain must also be stable. Preferably, they are also amplifiable, after incorporation into their DNA sequence of the modified base, analogous to the natural base.

 The "restriction -" character (restriction minus) is essential for the implementation of the preferred method according to the invention. It is this character which allows the introduction and maintenance of certain foreign nucleic acids in micro-Drganism. In the absence of this characteristic, the modified vectors introduced into the microorganism would be immediately hydrolyzed by those of restriction enzymes, whose specific role within the microorganism is to destroy the foreign nucleic acids introduced into it. this.

 Preferably also the strain has been made auxotrophic for the corresponding natural base. However, this auxotrophy could be replaced by any other mutation (or by any other biochemical means) having the effect of strengthening the capacity of the transformed microorganism to incorporate the modified base, for example to block metabolic access to thymidilate synthetase or to otherwise inhibit this enzyme.

 Preferably, the modified biologically incorporable base is a derivative of uracil or uridine, substituted in position 5 by a group containing at most two carbon atoms and, preferably, halogenated, and in that the microorganism is preferably auxotrophic for thymine or for the corresponding nucleosides or nucleotides. More particularly still, the uracil or uridine derivative is a halourouracil or a halourouridine, preferably bromouracil or bromo-uridine.

 In the latter cases, the microorganism used has mutations affecting at least some of the genes coding for the endogenous synthesis of thymine, under the control of thymidylate synthetase, in particular a mutation in the thyA locus.

 It is known that such clones can, in particular when they are mutants of E. coli, be selected in the presence of trimethoprim, according to the technique described by MILLER et al. (1972, "Experiments in Molecular Genetics, Ccld Spring Harbor Laboratory).

 An additional selection makes it possible to reduce the auxotrophy to a very low level, and at the same time allows an excellent final incorporation of halogenated uridine, in particular bromo-uridine (BrU). This selection is based on the observation that the bacterial metabolism is oriented towards the degradation of exogenous deoxyri bonucleotides, degradation which must be prevented.

All other catabolic mutations, in particular those associated with catabolic repression (journal ULLMANN DANCHIN, 1983, Adv. Cyc. Nue. Res. 15: 1-53) may also be used, advantageously in association with the mutations of which it is the question below and whose role will be to slow down or even suppress the catabolism of carbon strains. This additional selection will preferably involve the production of mutations in the genes controlling the degradation of deoxyribose-phosphates, in particular in the deo operon ( BACHMANN, KB, 1980, 44: 1-56, Microbiological
Reviews, and MILLER (1972), in particular in the deoB gene: which controls the synthesis of phosphopentomutase
EC 2.7.5.6, and / or in the deoC gene which controls the synthesis of deoxyribose-phosphate aldolase EC 4.1.2.4). Such a mutation is obtained by selecting from the thyA strain which requires a high level of exogenous thymine (50-100 micrograms per milliliter) clones growing at a low level (for example in culture media containing less than 10 micrograms / milliliter, especially around 1 microgram per milliliter maximum).

 As already mentioned above, preferred strains according to the invention consist of mutants little capable of catabolizing carbon sources, these mutants being in particular provided with mutations in the loci (cya) coding for the adenyl cyclase or in the locus (crp) coding for a cyclic AMP receptor or in both loci at the same time; this is useful to allow the presence inside the cell of a high level of phosphorylated desoryriboses, precursors of deoxyribonucleotides obtained by incorporation of exogenous bases.

As regards bacteria7, such mutants can be selected by taking advantage of the known resistance of such mutants to the antibiotic known under the brand name "MECILLINAM" (Laboratoires LEO) and to the virulent lambda phage. Such strains can be selected using the following operating conditions
100 microliters of a saturated culture of the initial strain are spread in the presence of a dis times higher number of a virulent lambda bacteriophage, on a Petri dish
containing Mac Conkey Maltose medium (MILLER 1972), supplemented with the minimum lethal concentration for
Antibiotic E. coli. This antibiotic, in fact, allows efficient selection of mutants lacking adenylate cyclase (cya) or cyclic AMP receptor (crp) (R. AONO, M. YAMAZAKI, G. TAMURA (1979),
J. Bact. 137: 839-845); in addition, the cya or crp strains are resistant to virulent lambda phage (review
ULLMANN and DANCHIN 1983) and since they do not ferment maltose, they appear in the form of white colonies on the Mac Conkey medium.

 In the preferred embodiments of the method according to the invention, the microorganisms used simultaneously comprise all of these mutations.

They will preferably also include, in addition to the mutations which have been mentioned, those which affect the phosphotransferase system (system dependent on phosphoenolpyruvate glycose: phosphotransferase: loci ptsHI crr in particular, but also associated systems, as well as other systems linked to the catabolic repression (ULLMANN DANCHIN review) The cya mutation can be considered as contributing strongly to the repression of catabolism.

 It should be noted that another essential, particularly important preferred characteristic of the strain used is the stability of its DNA. It is therefore necessary to select strains carrying mutations in the genes coding for exonucleases and endonucleases involved in DNA repair systems, for example mutations of the recB or recC type, or both in E. coli (exonuclease V); other mutations affecting both exonucleases and endonucleases, as well as the system for correcting reading errors at the replication level (SOS system) can also be used, in place of or in addition to those found in the strain described.

The different operations for realizing
be the above mutations, whether those
described in the literature or those that have been
recalled above (or others within the reach of the ex-
specialist in this technique) can
naturally be performed in any order,
subject to any opera incompatibilities
selection between them, as soon as they
are susceptible to mutual interference at the level
of the desired result. It goes without saying that the specialist
will be able to make the necessary choices regarding
to the most favorable order of selection operations
successive actions to be performed, depending on the nature of the
initial strain that he chooses to use.

Advantageously, we will proceed under the condi
following information, especially when recourse is
to the next initial strain known to be effective
with which it can be transformed and the absence
of restriction system of DNA: strain C600 SF 8
(CA.STRUHL, JR. CAMERON, RW. DAVIS (1976), PNAS 73:
1471-1475).

 It is treated so as to obtain a mutant little capable of effecting the catabolism of exogenous carbon sources. It is subjected to the selection operations under the conditions described above, in the presence of the virulent lambda phage and of "MECILLINAM" (4 micrograms / ml) and of maltose (1% by volume).

Cya or crp strains are resistant to virulent lambda phage and do not ferment maltose appearing as white colonies on the medium
Mac Conkey .. On the contrary, the colonies that use maltose during their fermentation are white.

A clone TP 6002, carrying the stable cya mutation, was kept. From this clone, an auxotrophic mutant for thymine (thyA) was isolated by selection in the presence of trimethoprim (according to the method described by MILLER 1972), which gives the stable strain TP 6030. An additional selection makes it possible to reduce the auxotrophy to a very low level, which allows at the same time an excellent final incorporation of BrU, by culture of this strain, in media increasingly depleted in thymine, until reaching a level of 1 microgram per milliliter maximum.

Several clones were obtained. The clone TP 6033 was subsequently used because of its stability.

 It should be noted that another essential characteristic of the strain is the stability of its DNA, due to the presence of the recBrecC mutations (exonuclease V) present in the strain C600 SF 8 chosen inter alia for this reason, and to the presence of mutations affecting catabolism (cya in particular).

The strain finally obtained can be cultivated in LB medium containing, for 1 liter, 10 grams of
Bacto tryptone and 5 grams of yeast extract "BACTO
YEAST EXTRACT ", both manufactured by DIFCO, and 10
grams of sodium chloride. It can be stored in the same medium additionally containing 8% by volume of dimethyl sulfoxide, mannitol at a final concentration of 0.1% by weight and optionally superoxide dismutase (SOD) obtained from beef blood, at a rate of 2 micrograms per milliliter (or for 3000 units per milligram of protein). In general, the thymine content provided by the LB medium is sufficient.

If necessary, it can be supplemented with 1 microgram of thymine / ml.

 The process for obtaining a cold probe according to the invention can also be used for the manufacture of probes in which it is no longer the thymine which is substituted, at least in part by a corresponding modified base, but a base separate. The application of the method according to the invention to the latter case however requires taking into account an additional constraint, namely an appropriate compartmentalisation between the ribonucleotides and the deoxyribonucleotides, within the cells in culture.

Indeed, thymine, ribonucleosides or ribonucleotides derived from thymine are not involved in the major pathways of cell metabolism. This is not the case for the other ribonucleosides. Endogenous interconversion between ribonucleotides and deoxyribonucleosides must be prevented, in which the base concerned would also be modified at the level of the corresponding base. Compartmentalisation, which is natural for thymine, must therefore be imposed genetically for the other bases. This is possible, for example, by carrying out mutations in the genes coding for ribonucleoside diphosphate reductase (EC 1.17.4.1.), In particular at the level of the nrdA and nrdB loci (BACHMANN loc. Cit).

The mutants obtained are then auxotrophic for the deoxyribonucleosides.

 The method according to the invention, as defined above for manufacturing the probes in vivo then implies the presence in the culture medium, not only of the corresponding modified base (in ~ free or combined state) , but also the other deoxyribonucleosides (natural or also modified) derived from the natural base.

 By way of example of the modified bases distinct from uracil, mention may be made of 5-bromocytosine, 8-bromoguanine and 8-bromo-adenine.

 The microorganisms used will also exhibit mutations similar to those which have been mentioned. The other mutations retain the same utility, even the same necessity.

 The preferred method according to the invention for manufacturing labeled probes has a double advantage in that it allows, from a vector DNA containing the insert insert complementary to the nucleotide sequence determined to be detected, chemical modification in vivo and simultaneous amplification of the probe. The techniques for transforming microorganisms, particularly mutated bacteria, by recombinant DNAs containing the insert to be labeled, are identical to those described in the literature, for example in "Molecular Cloning, A Laboratory Manual" by T. MAGNATISet Coll.

 When the recombinant DNA consists of a plasmid containing the insert to be labeled, it is advantageous to carry out the culture, in the presence of the modified base, preferably at a concentration of 0.5 to 500 and even more preferably of 1 to 50 micrograms per milliliter, the culture being preferably maintained for at least four generations and more. If possible.

The purification of the amplified probes in the microorganisms harvested at the end of the culture can be carried out in any known manner. It is advantageous to implement the technique described by
BIRNBOIM, HC and J. DOL "(1979-," A rapid alkaline extraction procedure for screening recombinant plasmid
DNA ", Nucleic Acids Res. 7: 1513).

The opening of the plasmid and, if necessary, the separation of the probe and the other parts of the plasmid also involve conventional techniques of genetic engineering, for example those described in "Molecular Cloning", "A Laboratory Manual "by T.MANIATIS,
EP FRITSCH and J. SAMBROOK (CSH).

 The implementation of the in vivo process allows substitutions, in particular of 25%, advantageously exceeding 75% of the content of natural bases of the probe treated.

It is even possible to obtain an almost total substitution of the natural base with the modified base, in particular by using high concentrations of the modified base in the culture medium of the microorganism.

Naturally, the probes according to the invention can also be constructed by using enzymatic or chemical synthesis methods in vitro. In particular, the so-called "nick translation" technique can be used, for example under the conditions described by RIGBY, PW and Coll. (1977,
J. Mol. Biol. 113, 237-251), but by substituting bromodeoxyuridine triphosphate (BdU) for thymidine triphosphate (TTP), at the same concentrations. The probe finally obtained contains a proportion of BdU
exceeding 25%. The probes obtained can be stored in a TE buffer solution (Tris.Hcl.

 10 mM, pH 8.0, 1 mM EDTA, pH 8.0).

The method according to the invention is not limited to the manufacture of probes in accordance with the present invention, that is to say in which the modified bases contain at most two carbon atoms. It can be extended to the production of modified probes, in which at least some of the natural bases are replaced by bases resulting from the modification of the preceding ones, insofar as said modified bases do not interfere with the pairing capacity
WATSON-CRICK of the modified nucleic acid which results from it with a natural complementary nucleic acid or containing similar modified bases. As such, the method according to the invention extends to the manufacture of probes, in which the bases modified include substitution groups, which can themselves contain more than two carbon atoms, for example bases modified by biotin. The essential condition which these modified bases must naturally also satisfy is that they are biologically incorporable in the sense which we have given above to this expression. As such, the bases, ribo- and deoxyribonucleosides and the ribo- and deoxyribonucleotides described in the European patent application mentioned above, since they also satisfy the above-mentioned condition.

 The method according to the invention is in fact applicable to any recombinant DNA, since it is capable of transforming a microorganism and more particularly an E. coli strain meeting the conditions which have already been indicated and which will be recalled below. -afterwards, whether or not this plasmid contains an insert as defined above. In general, the invention therefore provides a method of in vivo labeling of plasmids or other vectors, this labeling being capable of facilitating their subsequent recovery.

 The invention also relates to the microorganisms themselves which are particularly suitable for the production of probes in vivo, under the conditions which have been indicated.

 In particular, it relates to microorganisms, preferably of the bacteria type, such as E. ccli, characterized by a combination of mutations or properties - they are restriction -t ', - mutations affecting at least some of the genes, preferably all the genes, controlling the endogenous synthesis of one of the bases involved in the constitution of natural nucleosides, - mutations affecting at least some of the genes controlling the catabolism of carbon sources and intracellular deoxyriboses, - mutations affecting some at least of the genes, and preferably all the genes controlling the synthesis of nucleases affecting internal DNA.

 It also relates to the abovementioned microorganisms harboring a recombinant vector containing, where appropriate, an insert in the case where this vector or parts of it are intended to be used subsequently as a probe, these microorganisms also comprising a combination of mutations affecting simultaneously - at least some of the genes, preferably all the genes controlling the endogenous synthesis of one of the bases involved in the constitution of natural nucleosides, - at least some of the genes controlling the catabolism of the sources of carbon and intracellular deoxyriboses, - at least some of the genes and preferably all the genes controlling the synthesis of nucleases affecting internal DNA.

 Preferred microorganisms (transformed or not) are characterized by a combination of mutations simultaneously affecting the thyA, cya or crp loci or both, dec, recB, recC.

 The method according to the invention therefore provides a means allowing the easy renewal, and at relatively low cost, of all probes or more generally still of all recombinant DNAs which can be recognized by labeling techniques, for example according to the methods which have been envisaged, for example in European patent application No. 0063869-A3 and which preferred modes of implementation will be recalled below. Preferably, the detection is nevertheless carried out by means of polyclonal or, preferably, monoclonal antibodies. From this same point of view, the strains according to the invention transformed by a recombinant DNA (not yet labeled itself) can be maintained in culture or, if necessary, in the lyophilized state. When the strains in question are auxotrophic for one of the abovementioned bases, it is naturally advisable to maintain them (in the case where they are not in the lyophilized state) in a preservation medium containing low doses of the natural base.

They can therefore, on request, serve as a vector source modified by an insert - in the case of the cold probe - or unmodified, by contacting or culturing in a medium containing instead of the corresponding natural base a biologically incorporated modified base.

 The invention also relates to vector DNAs, in particular plasmids, cosmids or DNA of bacteriophages, in which- at least one of the bases is at least partially substituted by a modified base, in which the modification group comprises at most two carbon atoms, optionally halogenated, or consists of a halogen.

In the case where these recombinants constitute probes in the sense indicated above, that is to say that they are modified by an insert complementary to a determined sequence of nucleotides which is to be sought in biological samples, they can be implemented in all the applications which have been set out in the above-mentioned European patent application, that
either for applications, for example medical diagnosis, gene mapping, production of karyotypes, detection of genetic anomalies, identification or detection of specific microorganisms.

The vectors according to the invention can, like the "normal" vectors with regard to the constitution of the nucleotides entering their nucleic acids, be used as cloning vectors. They can be excised by restriction enzymes and recombined with other nucleic sequences, using the
classic endonucleases or ligases depending on the nature of the operation
performed, in other words be used in genetic engineering techniques.

 They can also be used as selection markers, thanks to the modified bases which they contain. In this regard, they are of a particularly interesting application when it comes to detecting those of cells (prokaryotes or eukaryotes) into which they have been introduced, if necessary with a particular insert whose expression in the cells is wanted.

This labeling is all the more interesting as the detection of the modified bases, in particular by antibodies selectively directed against the modified bases,
does not necessarily involve the prior extraction of nucleic acids from the transformed cells. Detection
tion can be performed on whole cells, even intact.

 Preferably, the antibodies used for detection will be monoclonal. They behave as if they penetrate inside cells and they can, in their turn, be recognized by other antibodies directed against them or by distinct affine molecules, these other antibodies or affine molecules carrying for example the group enzymatic or fluorescent allowing their detection by conventional methods.

 An example of the preparation of monoclonal antibodies directed against bromo-uridine is described below.

Monoclonal antibodies suitable for recognition
of the preferred modified nucleotide, in particular by the
iodouridine, can be prepared as shown below
after.

Halogenated nucleotides (BrUd, iodouridine) have
been coupled to proteins (albumin and ovalbumin),
in order to make them immunogenic. This coupling has been
carried out by a process which consists in oxidizing the ribose
nucleotide by sodium periodate; the functions
aldehydes thus created react in a second
time with amino groups of the protein
(ERLANGER and BEISER, PNAS US, 52, 68 (1964). The subs
titution was about 5 to 15 nucleo molecules
tides per protein molecule (support).

Mice from a cross between BALB / C
and C57BL / 6 were immunized with iodouridine neck
added to the emulsified ovalbumin in the substances
complete Freund's adjuvants at the rate of 200 micro
grams per mouse and per injection. The mice have
received two to three injections one month apart.

The spleen and popliteal cells of a
mouse having received a booster injection beforehand
were fused to murine myeloma SP20 by the intermediary
diary of polyethylene glycol according to the technique described by KOHLER and MILSTEIN, Nature, 256, 495 (1975) and modified by GALFRE et al., Nature, 266, 550 (1977).

The supernatants of the wells containing cell clones were tested by an immunoenzymatic method, albumin coupled with either BrUd or methyl uridine (thymidine analog) was fixed to polystyrene plates. The plates are incubated with the supernatants and then with a mouse anti-Ig coupled to beta-galactosidase. The clones recognizing only albumin-BrUd albumin-thymidine were selected, cloned and the cells injected into mice so as to obtain ascites rich in monoclonal antibodies. Antibodies were isolated from these ascites on a protein-A column coupled to Sepharose.

The purified antibodies were tested for their recognition only of BrUd and not of thymidine on nuclei of mammalian cells (fibroblasts) cultivated in the presence or not of BrdUd by an immunoenzymatic technique.

 A hybridoma secreting such antibodies was deposited on November 23, 1983 at the National Collection of Culture of Micro-Organisms of the Institut Pasteur de Paris (C.N.C.M.). He was assigned the number I-263.

The contacting of these monoclonal antibodies with the probe or the DNA-vector comprising bases
BrUd can be achieved by using classic techniques.

 This contact can in particular be carried out after immobilization of the single-stranded or double-stranded DNA (coming from the sample to be studied, for example a fibroblast) on a nitrocellulose filter (or any other suitable support). Advantageously, the revelation will then be carried out by an immunocytochemical method comprising: the incubation of the filters with murine anti-DNA monoclonal antibodies. washing, incubation with mouse anti-immunoglobulin antibodies labeled with peroxidase, washing, histochemical staining of the enzyme. This technique makes it possible to highlight up to 0.1 μg of DNA which has incorporated BrU, deposited on the nitrocellulose filter, while no coloration is observed with the corresponding natural DNA.

The results obtained indicate that the experimental conditions used make it possible to obtain effective detection sensitivities without any amplification system having been used. The absence of background noise makes it possible to envisage an amplification of sensitivity at least by a factor of 10.

Any known detection method can of course be used. The methods described in the articles whose references follow are particularly advantageous, in particular in relation to the following questions - Immunocytochemical detection of peroxidase coupled to antibodies present in cells
AVRAMEAS (Immunochem., 1969, vol. 6, p. 825) - Detection of uncoupled peroxidase, present in tissues
GRAHAM et al (Histochem. And Cytoehem., 1965, vol. 13, p. 150) - Use of beta-galactosidase as a marker in an enzyme immunoassay
GUESDON, THIERRY, AVRAMEAS (J. Allergy Clin. Immunol., 1978, vol. 61, p. 23).

 Additional information relating to the invention will also appear during the description of the conditions under which the various operations can be carried out. Of course, they are not limiting.

Use is made of the strain of E. ccli, and more particularly of the clone TP 6033, which has been previously transformed by any DNA-vector, for example by the techniques described in "Molecular
Cloning, A Laboratory Manual "by T. MAGNATIS et al., Insofar as these describe E. coli transformation procedures.

 This transformation was carried out on the above-mentioned strain in a culture medium containing the natural thymine.

BACTERIA CULTURE FOR INCORPORATION OF 5-BU (5-bromo-uridine)
The bacterium is cultivated in medium 63 B1 or minimum medium supplemented with glucose, casamino acids and adenine (0.1 mg / ml) and thymine (0.1 to 1 microgram / ml). The antibiotics necessary for the specific plasmid inserted into the strain are supplied to the culture medium.

 After inoculation of one of these media used for the preculture of the bacteria and culture at 370C up to an OD (optical density at 600 nm) of 1.2 to 2, an aliquot is removed and transferred (final OD of 0 , 1 to 0.3) in the medium concerned, without thymine but supplemented with 5-bromo-uracil (1 to 100 micrograms / ml) under the same conditions. The culture is maintained for at least two generations and the plasmid purified by conventional techniques (BIRNBOIM and DOLY. NAR 7, 1513).

For example, a culture of four generations, in the presence of 1 microgram / ml of 5-bromo-uracil, with a residual thymine concentration at most of 0.1 microgram / ml, makes it possible to obtain 25% substitution thymidine included in the incorporated plasmid.

I - DEPOSIT OF TARGET DNA ON FILTER
BA-85 nitrocellulose filters (SCHLEICHER and SCHUELL) were used.

 For deposits in small dot-blots, the DNA is diluted in TE or TBS (Tris HCl. 20; mM pH 7.8, 0.15 M NaCl) containing 1.5 mg / ml of DNA from sonic salmon sperm (DNA carrier), thermally denatured (1O00C for 5-10 minutes, then soaked in supercooled ice) before or not. The target DNA is predenatured using the same technique. The filters, air-dried, are then baked at 800C in a vacuum oven for 2 hours.

 The transfer from the agarose gels (electrophoretic separation) to the BA 85 filters is carried out according to the technique of THOMAS (P.S. 1980; DNAS 77, 5201-5205); after SOUTHERN (C.M. 1975, J. Mol. Biol.

98, 503-517).

II - PREHYBRIDATION
The prehybridization buffer is that described by
LEARY JL et al. 1983 ("Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose":
Bio-blots - PNAS 80, 4Q45-4049, according to WAHL and Coll.

1979 PNAS 76, 3683-3687), in which the sonicated herring sperm DNA is replaced by salmon sperm DNA at 250 micrograms / ml; the protein presaturation necessary for the immunoenzymological revelation is carried out jointly by adding normal serum (SN) from decomplemented sheep or goat (10% v / v) and bovine serum albumin (BSA) at 1%.

 Incubation takes place at 420C for 2 hours, at a rate of approximately 1 ml per 10 cm of filter; it is carried out in a "sealed bag" (scellobag) with slight agitation.

 The buffer is removed to be replaced by the hybridization buffer.

III - HYBRIDIZATION
The BU probe is denatured at 1000C (5-10 minutes), then suddenly cooled (ice). Incubation (2 to 9 hours) takes place as above with a hybridization buffer containing 45% deionized formamide, 250 micrograms / ml of DNA of sonicated salmon sperm (JL

LEARY et al., 1983 ,. '1Rapid and sensitive colorimetric method ... "Bio-blots. PNAS, 80, 4045-4049) and supplemented with normal serum / BSA (10% / 1%) as before.

The washes comply with JL LEARY et Coll. (1983, "Rapid and sensitive colorimetric method .." Bio-blots
PNAS, 80, 4045-4049) for a 115% hybridization of deionized formamide.

 The filters are air dried.

IV - DENATURATION OF HYBRID AREAS
Anti-BU antibodies better recognize single strand DNA than double strand, denaturation on a filter is carried out. The filters are soaked for 15 to 30 seconds in 0.5 M NaOh, 1.5 M NaCl, then 5 to 10 minutes in a neutralization solution (0.5 M Tris HCl, pH 8.0, 1.5 M NaCl) . The filters are then dried at room temperature.

V - IMMUNOENZYMOLOGICAL REVELATION
The filters are pre-wetted with TBS + TWEEN (0.1%) (TBS.T), then placed in - "scellobags".

The anti-BU antibody (mouse) is added to TBST with 10% of SN decomplemented from horse or sheep and
2 1% BSA at a rate of 1 ml per cm of filter. The incubation is carried out, with gentle shaking, for 2 hours at 370C or overnight at room temperature and at antibody concentrations from 1 microgram / ml to 20 micrograms / ml.

 Three 15-minute rinses with TBST are carried out (light stirring) at room temperature.

 A sheep antibody, directed against the mouse gamma globulin and coupled to peroxidase, is then added, under the same conditions (1 to 5 micrograms / ml) and left in contact with the filters for 2 hours at room temperature. The filters are then rinsed as above.

 The presence of the peroxidase is revealed by means of the substrate DAB (Diamino-benzidine) giving an insoluble product.

VI - RESULTS (figures expressed in pg of DNA present
on the filter). Sensitivity detection
0.5 to 5 pg of denatured DNA-BU (respectively mar
in vivo or by "Nick-translation") are obtained
by this method in "Dot-b lot".

 A detection of the order of 50 μg of DNA is ensured, after hybridization.

 These values are already, in the absence of any amplification of the signals or optimization of the systems, at the level of the systems, the most efficient.

The following strains have been deposited at the
CNCM November 22, 1983 - E. coli strain TP 6002 under the number I-259, - """TP6030""n I-260, -""" TP 6033 "" n I-261.

Claims (33)

 1 - Process for obtaining a vector DNA, in particular a plasmid, cosmid or bacteriophage DNA, in which at least one of the bases is at least partially substituted by a modified base which is biologically incorporated into the genomic DNA of a microorganism and not interfering with the WATSON-CRICK pairing capacity of the modified nucleic acid which results therefrom with a natural nucleic acid complementary or containing similar modified bases, characterized by the setting and maintaining in culture of the microorganism previously transformed by the corresponding DNA-vector in which the corresponding natural base is not modified, with a culture medium containing said modified base in replacement of the corresponding natural base, the aforementioned modified base being there, ie at free state, either in the combined state, in a nucleoside or nucleotide corresponding to said base, and by the subsequent recovery of the DNA-recombinant or DNA-vector from the dud culture it microorganism.  2 - Method for obtaining a cold probe for the detection of a determined sequence of nucleotides by hybridization and which itself comprises an insert comprising a sequence complementary to said sequence of nucleotides, characterized in that at least part bases of at least one of the types of nucleotides constituting its nucleic acid is replaced by modified bases recognizable by the polymerases, said modified base being biologically incorporable in the genomic DNA of a microorganism and not interfering with the WATSON-CRICK pairing capacity of the modified nucleic acid which results therefrom with a natural nucleic acid complementary or containing similar modified bases, characterized by setting and maintaining in culture the microorganism previously transformed by a probe in which the corresponding natural base is not modified, with a culture medium containing the modified base to replace the corresponding natural base spondant, the aforementioned modified base being there, either in the free state, or in the combined state, in a nucleoside or nucleotide corresponding to said base, and by the subsequent recovery of the DNA-recombinant or DNA-vector from the culture of said microorganism.  3 - Process according to claim 1 or claim 2, characterized in that the modified base comprises a group comprising at most two carbon atoms and, preferably, at least one halogen, or a group consisting of a halogen.  4 - Method according to any one of claims 1 to 3, characterized in that the microorganism is a mutant of bacteria, preferably E. coli.  5 - Process according to any one of claims 1 to 4, characterized in that the microorganism is or has been made auxotrophic for the natural base corresponding to the above-mentioned modified base.  6 - Process according to claim 5, characterized in that the modified base is a derivative of uracil or uridine, substituted in position 5 by a group comprising at most two atoms and, preferably, halogenated and in that the microorganism is auxotrophic for thymine or for the corresponding nucleosides.  7 - Process according to claim 6, characterized in that the modified base is a halourouracil or a halourouridine, preferably bromo-uracil or bromo-uridine.  8 - Process according to claim 6 or claim 7, characterized in that the microorganism has a mutation affecting the gene or genes coding for the endogenous synthesis of endogenous thymine, under the control of thymidylate synthetase, in particular a mutation in the thyA locus.  9 - Method according to any one of claims 1 to 5, characterized in that the biologically modified base is derived from a natural base distinct from thymidine or uracil or from a nucleoside corresponding to this distinct base, that the microorganism is auxotrophic for deoxyribonucleosides and in that the culture is carried out in the presence not only of the corresponding modified base (in the free or combined state), but also of deoxyribonucleosides derived from other natural bases.  10 -A method according to claim 9, characterized in that said microorganism comprises the genes coding for ribonucleoside diphosphate reductase, in particular in the locus nrda, or in the locus nrdB, or preferably in both loci at the same time.  11 - Method according to any one of claims 1 to 10, characterized in that the strain is a mutant little capable of effecting the catabolism of carbon sources, this mutant being in particular provided with mutations in the locus (cya) coding for adenylcyclase, or in the locus (crp) coding for a cyclic VAMP receptor or in both.  12 - Process according to any one of claims 1 to 11, characterized in that the aforementioned microorganism also comprises mutations in the genes affecting the synthesis of phosphotransferases, in particular in the ptsHI crr loci.  13 - Method according to any one of claims 1 to 12, characterized in that the aforesaid microorganism also comprises mutations in the operons controlling the degradation of the deoxyribose phosphates, in particular the operons (deo) and more particularly in the operon ( deoB) or in the operon (deoC) or in both operons at the same time.  14 - Method according to any one of claims 1 to 13, characterized by mutations in genes coding for exonucleases and endonucleases involved in DNA repair systems, such as mutations of the recB or recC type, or two at a time, especially when the microorganism used is E. coli.  15 - Microorganism, preferably of the bacteria type, such as E. coli, capable of being used in the process according to any one of claims 1 to 14, characterized by a combination of mutations or properties: - it is 1, restriction minus ", - mutations affecting at least some of the genes, preferably all genes, controlling the endogenous synthesis of one of the bases involved in the formation of natural nucleosides, - mutations affecting some at least of the genes controlling the catabolism of carbon sources and intracellular deoxyriboses, - mutations affecting at least some of the genes, and preferably all the genes controlling the synthesis of nucleases affecting internal DNA.  16 - Micro-organism according to claim 15, characterized by a combination of mutations simultaneously affecting the thyA, cya or crp loci or both, deo, recB, recC.  17 - Microorganism hosting a recombinant vector containing and also comprising a combination of mutations simultaneously affecting - at least some of the genes, preferably all the genes controlling the endogenous synthesis of one of the bases involved in the constitution of natural nucleosides, - at least some of the genes controlling the catabolism of carbon sources and intracellular deoxyriboses, - at least some of the genes and preferably all of the genes control the synthesis of nucleases affecting internal DNA.  18 - Microorganism according to claim 16 or claim 17, characterized by a capacity for growth in a culture medium containing less than 10 micro grams / milliliter, preferably of the order of 1 microgram / milliliter of exogenous thymine.  19 - Microorganism according to claim 16 or claim 17, characterized by a combination of mutations simultaneously affecting a locus controlling the endogenous synthesis of a natural base distinct from thymine, and the loci cya, crp, deoB, deoC, recBc , nrdA and nrdB.  20 - recombinant DNA, characterized in that at least part of the bases of at least one of the types of nucleotides constituting its nucleic acid is replaced by modified bases comprising at most two carbon atoms.  21 - recombinant DNA forming a cold probe for the detection by hybridization of a determined sequence of nucleotides and itself comprising an insert comprising a sequence complementary to said nucleotide sequence, characterized in that at least part of the bases of at least one of the types of nucleotides constituting its nucleic acid is replaced by modified bases recognizable by the polymerases and comprising a group comprising at most two carbon atoms.  22 - DNA-recombinant according to claim 1, characterized in that this group comprises at least one halogen or is constituted by a halogen.  23 - DNA-recombinant according to any one of claims 20 to 22, characterized in that it is homologous to a replicable vector in a microorganism, such as phage DNA, or preferably plasmid, modified by the above insert.  24 - DNA-recombinant according to any one of claims 20 to 23, characterized in that the modified bases are chosen from those which are capable of being incorporated biologically into the genomic DNA of a microorganism, in particular of E.coli.  25 - DNA-recombinant according to any one of claims 20 to 24, characterized in that the modified bases are derived from natural nucleotide bases the modification of which resides in a substitution with the aforementioned group1 in position 5, when it is a question of a pyrimidine base, and in position 7 or in position 8, or in both positions at the same time, when it is a deazapurine or purine base.  26 - DNA-recombinant according to any one of claims 20 to 25, characterized in that the modified bases are 5-halo-uracil groups, preferably 5-bromo-uracil.  27 - DNA-recombinant according to any one of claims 20 to 26, characterized by the structure

  in which - each of the letters B, B ', B "... represents a purine, 7-deazapurine or pyrimidine group, each of these groups B, B ', B "... being covalently linked to the corresponding sugar group in the C1 position thereof, either at its own N9 position, for the purine or 7-deazapurine groups, or at the level of its own position N1 for the pyrimidine groups, - A represents a group of modifications of B, B ', B "..., as the case may be, consisting of a substitution group comprising at most two carbon atoms and, preferably, being in addition halogenated, this group A being directly linked to the heterocyle of the nitrogenous base, either in position 5, when B, B ', B "... are pyrimidine groups, either in position 7, or in position 8 , or in both positions at the same time, when B, B ', B "... are deazapurine or purine groups, - z represents -H or -OH and - m and n represent whole numbers from O to 100,000 respectively , - p is an integer from 1 to 75,000, it being understood that the sum m + n + p is at least equal to 2, preferably at least equal to 100.  28 - DNA-recombinant according to claim 27, characterized in that the groups B - A are 5-halogeno-uracil groups, preferably 5-bromo-uracil  29 - DNA- recombiannt according to any one of claims 20 to 28, characterized in that the substitutions of bases modified with natural bases are in a proportion of at least 25%, preferably exceeding 75% of the total content of bases corresponding natural of said probe;  30 - hybrid DNA of which one of the strands consists of a nucleic acid containing the determined sequence of nucleotides defined in claim 21 and the other strand of which consists of the cold probe according to claim 21 considered either alone or in combination with any one of claims 22 to 29, or by a part of said probe nevertheless comprising the above insert.  31 - Complex formed between, on the one hand, a probe according to any one of claims 20 to 29 or a Hybrid DNA according to claim 30 and, on the other hand, an antibody selective for the above-mentioned modified bases, the antibody being either labeled itself or capable of being recognized by another labeled antibody or other labeled affine molecule.  32 - Method for detecting a nucleic acid containing the above-mentioned determined nucleotide sequence, characterized by bringing this nucleic acid into contact with the cold probe according to claim 21, either alone or in combination with any one of claims 22 to 29 under conditions permitting their respective prior denaturations, if necessary, and their mutual hybridization, the separation of the excess probe not having intervened in said mutual hybridization, and the detection, direct or indirect of the hy- flange possibly formed, in particular by reaction with an antibody directed against the modified bases of the probe, the latter possibly being able in turn to be detected by another antibody or another molecule affine for the first antibody.  33 - Method for labeling or detecting cells into which a vector containing a particular insert has been introduced, the expression of which in said cells is sought, characterized in that the above vector containing the above insert conforms to that obtained by the process according to any one  of claims 1 to 14 and in that the detection is carried out by bringing the cells thus transformed into contact with an antibody directed against the modified bases of the above vector.