WO2003046217A2

WO2003046217A2 - Universal dna chip, method for making same, and uses thereof

Info

Publication number: WO2003046217A2
Application number: PCT/FR2002/004005
Authority: WO
Inventors: Pierre Taberlet
Original assignee: Commissariat A L'energie Atomique; Centre National De La Recherche Scientifique; Universite Joseph Fourier
Priority date: 2001-11-26
Filing date: 2002-11-22
Publication date: 2003-06-05
Also published as: WO2003046217A3; AU2002364792A1; FR2832730A1

Abstract

The invention concerns a universal deoxyribonucleic acid (DNA) chip, a method for making a genetic print with said chip, and various uses of said chip. The inventive DNA chip is a hybridization chip of a nucleic fragment collection extracted from an organism comprising a support whereon is fixed an ordered array of nucleic acid probes, said probes including first and second sequence parts, the first sequence being constant from one probe to another and at least complementary to the remainder of a sequence of a specific restriction site, and the second part, adjacent to said first sequence part being variable from one probe to another and consisting of specific combinations of nitrogenous bases selected such that the ordered array of probes comprises the probes capable of specific hybridization with the various fragments of said collection nucleic acid. The inventive chip enables to make a genetic print of the organism, and can be useful for identifying the sex, race, variety, species and the like of an individual from his DNA sample.

Description

UNIVERSAL DNA CHIP, MANUFACTURING METHOD THEREOF, AND CHIP USES

TECHNICAL FIELD The present invention relates to a universal deoxyribonucleic acid (DNA) chip, to a process for manufacturing said universal chip, as well as to various uses of said chip.

By universal DNA chip is meant a DNA chip which, thanks to its manufacturing process according to the present invention, makes it possible to identify very easily and reproducibly any living being provided with DNA. In fact, the present invention makes it possible to easily and reproducibly obtain a precise genetic imprint of a given individual.

The present invention applies both to animals and to plants, it makes it possible, for example, to identify the sex, race, variety, species, etc. of an individual or of a derivative product derived from this individual. from his DNA. It also applies to microorganisms such as bacteria, yeasts, algae, etc.

It finds for example an application in the fight against fraud on names or appellations in the food sector, for example to characterize varieties of plants, for example grapes, or for the purposes of individual traceability of animals, for example beef. It also finds, for example, an application in judicial inquiries, in forensic medicine, in providing a powerful, quick and easy-to-use tool for identifying an individual.

It also finds, for example, an application in the identification of genes involved in detectable genetic characters such as diseases of genetic origin. Indeed, a comparative analysis of several DNA chips manufactured according to the method of the present invention from several individuals carrying and not carrying a detectable genetic character can make it possible to locate on the chip DNA fragments linked to this character. detectable, and thereby even isolate and identify the genes involved in this or these character (s).

There is currently no biochip or manufacturing technique thereof having as large a potential as that of the present invention.

PRIOR ART Due to the interest shown, in particular by medicine, research and the pharmaceutical industries, in the detection and identification of the genes responsible for genetic diseases, due to the current crisis of consumer confidence regarding meats , in particular cattle, because of the growing number of frauds concerning appellations of origin, due to the interest shown, in particular by the courts, in a precise and rapid proof tool for identifying an individual or a product derived from a biological sample thereof, many research has been carried out and is being carried out to develop reliable and rapid tools for the identification of an organism or an extract of it and in particular tools using DNA. Genetic information is stored in every cell of living things in the form of long molecules of deoxyribonucleic acid (DNA) that make up the genome. It consists of a chain of four nitrogenous bases called nucleotides which are adenine, cytosine, guanine and thymine noted respectively A, C, G and T.

The basic premise regarding methods using DNA is that it is possible to identify an individual by his DNA. Indeed, apart from real twins or clones, each individual is genetically unique due to the mixing of chromosomes at the time of reproduction. By combining all the possible differences throughout the genome, given the number of nitrogenous bases, it appears that an infinity of combinations are possible, which makes each individual unique from a genetic point of view.

Many methods have been developed to fabricate genetic fingerprints to highlight this uniqueness of each individual. They can be classified into two categories: specific methods and universal methods.

The specific methods require significant and different technical developments for each species studied. The analysis of microsatellites polymorphism or SNPs ("Single Nucleotide Polymorphisms") are specific methods which unfortunately require knowledge about the genome sequence before their implementation.

Universal methods require little development, and can be applied without prior knowledge of the genome. These are the following methods: multilocus minisatellite probes, RAPD ("Randomly Amplified Polymorphic DNA" or randomly amplified polymorphic DNA), ISSR ("Inter-Simple Sequence Repeat"), AFLP ("Amplified Fragment Length Polymorphism" and more recently, the DArT method ("Diversity Array Technology" or density network technology). In the following text, the references in square brackets “[]” refer to the annexed list of references.

The method using multilocus mini-satellite probes was first described by Alec JEFFREYS, for example in documents [1] and [2]. It does not require amplification and comprises the following stages: (i) extraction of genomic DNA, (ii) digestion with a restriction enzyme, (iii) migration on agarose gel, (iv) hybridization with a probe s 'hybridizing with multilocus mini-satellite sequences (southern blot method). This approach has many drawbacks, in particular it is difficult to automate, it requires a lot of manpower and has many limitations, for example the fact that only profiles having migrated over the same gel. Today this method is less and less used.

The RAPD method was developed in the early 1990s by WILLIAMS et al. and by WELSH and McCLELLAND. It is described for example in documents [3] to [8]. It consists in amplifying by polymerase chain reaction (PCR) genomic DNA using arbitrary primers, generally very short of approximately 10 base pairs. A single primer is used in the amplification reaction. The fragments resulting from the amplification are then analyzed on agarose gel. This technique, although widely used, however requires the use of numerous amplifications by samples and proves to be difficult to implement due to problems of reproducibility.

The ISSR technique is described for example by ZIETKIEWICZ et al., 1994; KANETY et al., 1995 in documents [9] and [10]. It is similar to RAPDs in that it also uses arbitrary primers, but these primers are specific. They correspond to microsatellite sequences, with some additional bases on the 3 'side. After extraction, the genomic DNA is amplified, using an ISSR primer, then the fragments are visualized on polyacrylamide gels. The level of polymorphism is not always sufficient at the intraspecific level, and the analysis of gels is sometimes complex.

The AFLP technique developed by the company KeyGene produces a real genetic imprint. It is described for example by VOS et al., 1995 in the document [11] and by AJMONE-MARSAN P. et al., 1998 in document [12]. Here, after extraction, the DNA is digested with two restriction enzymes, one recognizing six base pairs and cutting infrequently, for example ECO RI, and the other recognizing four base pairs and cutting much more frequently, by example MSE I. In the presence of two adapters specific for these two restriction enzymes, the next step is the ligation of these adapters at the end of the restriction fragments. Next, amplification by the PCR method occurs in two stages: preselective amplification, where primers having a base pair extension on the 3 ′ side are used, so that only a part- only of the fragments restriction with adapters will be amplified, followed by selective amplification, where the product of the preselective amplification is again amplified, but this time with primers having an extension of two additional base pairs on the 3 'side. Thus, the genome is simplified so as to obtain only a hundred amplified fragments, which are then analyzed on polyacrylamide gel. This technique has the disadvantage that for each individual analyzed, it is necessary to use several pairs of primers with different extensions in order to obtain a reliable genetic fingerprint.

The DArT method, published very recently by JACCOUD et al., 2001 in document [13], uses the "microarray-to-DNA" approach to reveal polymorphisms. In this method, the DNA fragments of an organism deposited on the microarrays are produced in the following way: after digestion with a restriction enzyme such as Eco RI, Pst I, and Msp I, adapters are attached by ligation at each end of the restriction fragments. These fragments are then amplified by PCR using a single primer which corresponds to the sequence of the adapter, the sequence of the restriction site, and from 1 to 3 selective bases. These PCR fragments are then cloned, then deposited on microarrays

("Microarrays"). The analysis of the diversity is carried out by hybridization of the DNA of a sample on these micromatrices after a preparation similar to that of the deposited DNAs, that is to say digestion-ligation then amplification. The major progress therefore consists in avoiding electrophoresis. However, the choice of probes to deposit is different for each different species. The proposed method is therefore not universal and difficult to implement.

Many techniques have therefore been developed in recent years, but they all still have numerous drawbacks such as those mentioned above, in particular that they require several amplifications and several migrations to obtain a genetic fingerprint, with the exception of the DarT method. gel before providing a result. This leads to problems of implementation, reproducibility, precision, cost etc. In addition, these experiments are difficult to automate, especially at the level of gel analysis.

In addition, none of the solutions proposed so far is truly "universal" in particular in the aforementioned applications, and they require knowledge of the genome sequence before their implementation.

STATEMENT OF THE INVENTION

The present invention specifically aims to provide a universal DNA chip overcoming the aforementioned drawbacks, a method of manufacturing this universal chip making it possible, for example, to obtain, from a single amplification, a genetic fingerprint based on thousands of markers, and uses of this chip.

The universal DNA chip of the present invention is a hybridization chip of a collection of single-stranded fragments of a deoxyribonucleic acid originating from an organism, said collection of fragments being obtained in particular by digestion of said deoxyribonucleic acid with at least one enzyme restriction having a determined restriction site, each fragment comprising a sequence residue of said restriction site and, adjacent to said sequence residue, an internal sequence specific to each fragment; said chip comprising a support on which is fixed an ordered array of nucleic acid probes, said probes comprising a first and a second part of sequence, the first part of sequence being constant from one probe to another and at least complementary to the rest of the sequence of the determined restriction site, and the second part of sequence, contiguous with said first part of sequence, being variable from probe to other and made up of determined combinations of nitrogen bases chosen so that the ordered array of probes includes the probes capable of hybridizing specifically to the different nucleic acid fragments of said collection.

The term "probe (s)" refers to the sequences of deoxyribonucleic acid attached to the support to form the array of probes according to the present invention. The term "chip" refers to the array of probes and support, the probes being fixed to the support in the form of a matrix.

The term “fragment (s)” refers to the fragments obtained in particular by digestion of the DNA extracted from the organism with at least one restriction enzyme. Indeed, the extracted DNA can undergo according to the present invention other modifications than the enzymatic digestion before being hybridized to the probes of the chip of the present invention. These other changes are discussed below. The chip carrier according to the present invention can be any of the carriers used for the manufacture of DNA biochips known to those skilled in the art. Such supports are described, for example, in application EP-1141391 filed by the COMMISSARIAT A L'ENERGIE ATOMIQUE (France). The methods of fixing the probes to the support can be those which are conventionally used in the technique of DNA biochips, for example that described in the aforementioned document.

The supports which can be used according to the invention are for example supports made of glass, silicon, polyacrylamide gel, polymer (see American patent No. 5,919,523), plastic (microwell plates). These can be nylon membranes.

In general, the support is treated to provide attachment or grafting surfaces for the biological molecules. This surface treatment ensures the formation of chemical functions which are generally hydroxyl functions. These functions make it possible to carry out the subsequent chemical steps of grafting the probes. In addition, the density of the sites available at this stage will determine the final density of biological probes on the support.

For glass or silicon supports covered with an oxide layer, chemical cleaning of the surface makes it possible to give it hydroxyl groups, SiO ₂ molecules giving the surface SiOH. Cleaning can be carried out under basic conditions (using soda or ammonia) or under acidic conditions (using hydrochloric acid, sulfochromic acid or sulfo-oxygenated acid). Measurements made by the drop angle method make it possible to characterize the efficiency of the transformation of the surface state of the support. The effectiveness of the treatment on the immobilization of the probes is characterized by hybridization with complementary targets. Fluorescence observations show that the more hydrophilic the surface of the support, the better the immobilization of the probes, which makes it possible to obtain a greater density of probes on the support. Preferably, the support is a solid support having a surface for the immobilization of oligonucleotides, said surface being the surface of a material chosen from Hf0 ₂ , Ti0 ₂ , Ta ₂ 0 ₅ , Zr0 ₂ and a mixture comprising at least one of these materials, said surface having undergone a treatment to make it hydrophilic.

Advantageously, the material is in the form of a layer deposited on a substrate. This layer can have a thickness of between a few nanometers and a micrometer. The substrate can be a substrate chosen from glass, plastic and semiconductor substrates, for example silicon. The material having this surface for immobilizing oligonucleotides may for example be a mixture comprising Si0 ₂ .

The preparation of the support advantageously comprises the steps of supplying said substrate, of depositing on the substrate a layer of a material chosen from Hf0 ₂ , Ti0 ₂ , Ta ₂ 0 ₅ , Zr0 ₂ and a mixture comprising at least one of these materials, and treatment of the free face of said layer to make it hydrophilic. The deposition step may for example consist in depositing a layer of material with a thickness of between a few nanometers and a micrometer. The step of supplying the substrate may consist in providing a substrate such as those mentioned above. The deposition step can consist in depositing a material comprising Si0 ₂ .

The deposition step can for example implement a deposition method chosen from evaporation under vacuum, atomization by ion beam, radio-frequency sputtering, magnetron sputtering, vapor phase deposition of atomic layers ( ALCVD) and the sol-gel deposit.

The step of treating the free face of the deposited layer may consist in cleaning the layer with a basic solution or with an acid solution.

The method may include an additional step consisting in structuring the free face of said layer.

This structuring step can implement a technique chosen from dry etching, wet etching and "lift-off".

According to the invention, the constitution of the collection of single-stranded fragments of the organism's deoxyribonucleic acid therefore determines the constitution of the chip. Indeed, the probes fixed on the support to form the chip all have a common part, called "first constant part from one probe to another", which is at least complementary to the rest of the sequence of the determined restriction site of the restriction enzyme used to constitute the collection of fragments. This complementarity must lead to specific and stable hybridization of the fragments of the collection on the probes of the chip. Restriction enzymes have the property of cutting double-stranded DNA molecules at the level of very specific nucleotide sequences, called restriction sites, characterized by a symmetry of the nitrogen bases at the cut-off point. Since these sites are only present in a limited number in DNA molecules, the cut points are limited, and therefore the number of DNA fragments obtained after cutting by the restriction enzyme. Once the enzyme has recognized its restriction sequences on the DNA to be cut, it cuts the DNA at the level of these sequences, leaving each side of the cut, or only one side for certain restriction enzymes which cut DNA before or after the restriction site, a sequence residue from said restriction site. This sequence rest therefore has a known sequence for a known restriction enzyme.

The inventors have used this property of restriction enzymes to carry out the present invention.

Indeed, the first part of constant sequence from one probe to another is synthesized so as to be at least complementary to the rest of the restriction site of the enzyme used to digest the DNA extracted from the organism, it that is to say to hybridize to said rest of the restriction site present on each fragment of the collection. For example, if the rest of the restriction site sequence present on each fragment has the sequence 5 ′ AATTC 3 ′, the constant part from one probe to another on the chip of the present invention should comprise the complementary sequence 3 ′ TTAAG 5 '. The restriction enzymes usable in the present invention and their restriction site are set out in more detail below in connection with the description of the method of making a genetic fingerprint of the present invention.

When the rest of the restriction site sequence is too short and the hybridization of this rest with its complementary sequence made and present on the chip does not seem to be able to offer sufficiently stable and specific hybridization, according to the present invention, an adapter oligonucleotide having a known sequence can be linked to said rest of the restriction site on each fragment. The fabrication, function, and attachment of the adapter are discussed in more detail below.

Thus, according to a particular embodiment of the present invention, said collection of single-stranded fragments of DNA originating from the organism can also be obtained in particular by ligation of an adapter oligonucleotide with determined sequence at the ends of the fragments of said deoxyribonucleic acid. of the organism obtained by digestion with the determined restriction enzyme.

In relation to this particular embodiment of the present invention, the first part of the sequence which is constant from one probe to another on the chip is manufactured in such a way that it is complementary to the rest of the sequence of the site. restriction of the restriction enzyme used and to said adapter oligonucleotide of determined sequence. For example, if the rest of the restriction site has the sequence 5'-AATTC-3 'and if a sequence adapter 3'-ATGG-5' is linked to said remain so as to obtain fragments 5 '-TACCAATTCXXXXXX ... -3 ', X representing the nitrogen bases of the internal sequence proper to each fragment, the first constant part from one probe to the other of the probes fixed on the support to form the chip of the present invention has the complementary sequence 3' -ATGGTTAAG-5 '. This first part of the probe, common to all the probes will therefore hybridize specifically to the 5 '-TACCAATTCXXXXXX ...- 3' fragments, mentioned above.

The constant part is then a sequence of nitrogenous bases complementary to the rest of the restriction site and to the adapter.

This embodiment of the present invention makes it possible to obtain a more stable hybridization of the fragments of the collection of DNA fragments on the chip, and greater specificity of the chip for the fragments obtained with the restriction enzyme.

For example, according to the present invention, when the restriction enzyme used to prepare the collection of fragments is EcoRI, the adapter oligonucleotide may be a sequence complementary to one or to a part of one of the following two oligonucleotides:

5 '-CTCGTAGACTGCGTACC-3'; and 5 '-AATTGGTACGCAGTCTAC-3'.

The adapter is not necessarily necessary if. the rest of the restriction site sequence is long enough for specific and stable hybridization on the chip of the present invention. A length is considered sufficient if it includes at least about six base pairs. After digestion of the DNA originating from the organism with the restriction enzyme, the fragments obtained therefore comprise part of their sequence which is a residue of the restriction site of the determined restriction enzyme, and part of their sequence which is specific to each fragment and which depends specifically on the organism and on the place of cut by the restriction enzyme.

Considering that the DNA of an organism is made up of a chain of the four nitrogenous bases which are adenine, cytosine, guanine and thymine noted respectively A, C, G and T, the second part of sequence is made variable from one probe to another and made up of determined combinations of nitrogen bases chosen so that the set of probes constituted by the ordered array of probes includes the probes capable of hybridizing specifically to the different nucleic acid fragments from the collection of fragments.

The number of nitrogen bases constituting each variable part of the probes is chosen, according to the invention, so that the probe complementary to a fragment of the collection, also provided with the sequence complementary to the rest of the restriction site of said fragment, can specifically recognize and stably hybridize to this fragment. The number of probes or pads of the chip of the present invention therefore depends on the chosen number of nitrogen bases constituting the variable sequence. The greater the chosen number of nitrogen bases constituting the variable sequence, the greater the number of plots will be so that, in accordance with the present invention, the different possible combinations of nitrogen bases necessary for a specific hybridization of all the fragments of the collection fragments are present.

For example, a variable sequence of n nitrogen bases, n being an integer which can range for example from 4 to 15, chosen from A, C, G and T leads to 4 ⁿ possible combinations, that is to say also to 4 ⁿ , variable sequences possible. Table I below gives non-limiting examples of the number of probes to represent all the possible combinations of bases chosen from A, C, G and T.

Table I

According to the present invention, each second part of variable sequence from one probe to another can for example have a constant length chosen from 4 to 12 nitrogenous bases, for example from 4 to 10 nitrogenous bases. Advantageously, the length of the variable sequence is 6 nitrogenous bases taking into account current techniques and the cost of manufacturing DNA chips. According to the present invention, the nitrogenous bases can be those presented in table (I) above.

The chip of the present invention can include all the probes representing all the possible combinations taking into account the number and the type of bases constituting the variable sequence according to the present invention or only a part of these probes. It suffices, according to the present invention, that the probe array comprises the probes capable of hybridizing specifically to the different fragments of the collection, or even to only a part of said fragments, provided that the genetic fingerprint formed from the collection of fragments makes it possible to identify the organism from which the DNA or a genetic character, or marker, determined has been extracted.

This reduction in the number of probes on the chip makes it possible to reduce the manufacturing price of the chip, for a given organism, while retaining its effectiveness in identifying said organism. In fact, according to the present invention, - after manufacturing a first genetic fingerprint of a determined organism with a determined restriction enzyme, on a chip according to the present invention comprising all the possible combinations of the variable part of the probes, advantageously, the number of probes present on the chip can be judiciously reduced for the manufacture of other chips intended for manufacturing imprints of the same organism, from the same determined restriction enzyme, to those necessary and sufficient for the identification of said organism.

Likewise, the number of probes can be reduced on a chip of the present invention for the identification of a detectable genetic characteristic, to those necessary and sufficient for the identification of said characteristic. These may, for example, be genetic characteristics allowing identification of sex, race, variety, species, etc. an individual or a derivative product derived from this individual from its DNA. These genetic characteristics are also called markers. By detectable, is meant detectable with the chip of the present invention, whether or not in relation to characteristics directly detectable on the body visually, and / or by biochemical analysis, etc.

The chip of the present invention is therefore universal when it includes all the possible combinations of the variable part of its probes because it makes it possible to form a specific genetic imprint from the DNA of any organism.

The chip of the present invention is specific to an organism when the number of probes for detection has been reduced to keep only those which are necessary and sufficient to identify said organism. Of course also, certain possible combinations can be eliminated due to deviations from the standard hybridization conditions set out below on the chip of the present invention. This is the case for example for combinations too rich in A / T OR in C / G. This problem is known to those skilled in the art

It is the combination of the variable part of the probes of the chip and the ordered matrix of the probes which will make it possible to form a specific imprint of an organism. Indeed the variable part allows. specific hybridization with the different internal sequences of the different fragments of the collection, and due to the arrangement in an ordered matrix of the different probes a true genetic imprint of the organism can be revealed.

By ordered array of probes is meant herein a matrix consisting of probes arranged in a determined manner with respect to their part of variable sequence, and reproducible identically so as to be able to reproduce the same matrix with the same determined arrangement of probes in look of their variable part. By matrix is meant any geometrical arrangement or not of the probes on the support provided that the matrix makes it possible to distinguish the hybridized probes from the probes not hybridized on this support when the chip is used to make a genetic imprint. It can be, for example, a matrix with m columns and p rows, m and p being whole numbers, a matrix formed by a arrangement of probes in a snail or in concentric circles, etc.

The fact that the array of probes is ordered in accordance with the present invention makes it possible to be able to compare different genetic fingerprints, manufactured with the chip of the present invention, from different DNA extracts, prepared with an identical restriction enzyme for these different extracts, and to objectively conclude the identity or non-identity of the fingerprints. The process for manufacturing the imprints of the present invention is set out below.

Each DNA fragment of the collection of fragments having its complementary probe on the chip of the present invention, bringing the collection of fragments into contact with the chip comprising its ordered probe matrix, under conditions suitable for hybridization, provides therefore a specific genetic imprint of said organism. The inventors have noted that a universal chip according to the present invention with a matrix of 10,000 probes or pads is sufficient to guarantee reliable individual identification. With such a chip, it is possible to analyze according to the present invention in a single experiment several hundred polymorphic markers in any species.

The present invention also provides a method for manufacturing a genetic fingerprint of an organism comprising the following steps: extraction of double-stranded DNA from said organism, determination of the concentration of double-stranded DNA extracted and, if necessary, dilution of the extracted DNA to a concentration of 1 to 20 ng / μl, digestion of the extracted DNA before or after dilution with means of at least one restriction enzyme having a restriction site determined so as to obtain double-stranded fragments comprising a sequence residue of said restriction site, and, adjacent to said sequence residue, an internal sequence specific to each fragment; optionally ligation of a double-stranded adapter oligonucleotide with a determined sequence at the ends of the double-stranded fragments obtained by digestion of said DNA to obtain suitable double-stranded fragments, amplification of the double-stranded fragments, optionally linked to the double-stranded adapter oligonucleotide, by a polymerization chain reaction using a primer complementary to said rest of the sequence of said restriction site and optionally to the double-stranded adapter oligonucleotide, labeling of the double-stranded fragments, optionally linked to the double-stranded adapter oligonucleotide, during or after their amplification, using a marker, denaturation of the labeled double-stranded fragments, possibly linked to the double-stranded adapter oligonucleotide, so as to obtain a collection of single-stranded DNA fragments coming from the organism possibly linked to the single-stranded adapter oligonucleotide, hybridization of said collection of fragments on a chip according to the present invention, - rinsing of the chip to eliminate in particular the non-hybrid fragments and drying.

A reading of the chip can be carried out by a means of detecting the marker used.

When the fragments of the collection are linked to the single-stranded adapter oligonucleotide, the hybridization step is carried out on a chip according to the present invention in which the first part of the sequence, which is constant from one probe to the other of the chip, is complementary to the rest of the restriction site sequence of the restriction enzyme used and to the adapter oligonucleotide with determined sequence.

The organism is a DNA organism, animal, plant, insect or microorganism.

According to the present invention, it is preferable that the DNA is of good quality, that is to say not too degraded and with may or no inhibitor (s) of restriction enzymes in order to be able to produce a usable genetic fingerprint. The extraction of DNA from the organism can be carried out by conventional methods of extracting DNA from an organism. By way of nonlimiting example, the extraction methods described in the work by Sambrook J, Firtsch EF, Maniatis T., 1989 Molecular cloning: a laboratory manual 2 ^nà edition, New-York: Cold Spring Harbor Laboratory Press, cold Spring Harbor, New-YorK, can be used.

According to the invention, The extractions can be carried out for example with the QIAGEN (trademark) extraction kits, for example QIAamp Blood Extraction Kit (registered trademark) for blood, QIAamp Tissue Extraction Kit (registered trademark) for tissues , or Dneasy Plant Mini Kit (registered trademark) for plants, following the manufacturer's protocols. According to the invention, the determination of the concentration of the double-stranded DNA extracted can be carried out by the conventional methods for assaying the DNA in an extract. For example, it can be determined by fluorimetry with PicoGreen (registered trademark) in DNA QUANTIFICATION KIT (trademark) ("Molecular Probes").

A few nanograms of DNA are sufficient to apply the method of the present invention. Also, when the DNA extract has a concentration greater than 20 ng / μl, advantageously, according to the invention, in order to be able to produce a “legible” genetic fingerprint, which does not saturate the chip of the present invention, the concentration extract can be reduced to a concentration of 1 to 20 ng / μl, for example from 5 to 15 ng / μl, for example to 10 ng / μl (nanograms per microliter).

Before or after dilution, if necessary, the DNA is "digested" with at least one restriction enzyme to obtain fragments of this DNA. The main objectives of the digestion step of the process of the present invention with the restriction enzyme are to provide a reasonable number of fragments of the extracted DNA making it possible to make a reproducible fingerprint, and that the fragments provided show residues of restriction site sequence of sufficient length to allow specific hybridization with the constant part of the probes complementary with said restriction site sequence residues, and optionally the attachment of an adapter.

Many restriction enzymes have been identified to date, as well as their restriction sequence. A person skilled in the art, on reading the present description, can easily choose the most suitable restriction enzyme for the application which he will carry out of the present invention from the restriction enzymes identified, and from those which will be identified in the future. The literature on restriction enzymes generally provides for each enzyme its specific restriction site.

Restriction enzymes which can be used according to the present invention can be found, for example, in catalogs from suppliers such as Roche Molecular Biochemicals, New England Biolabs, Stratagene etc.

As nonlimiting examples of restriction enzymes which can be used according to the present invention, mention may be made of EcoRI extracted from E. Coli, HaellI extracted from Haemophilus aegyptus and PstI extracted from Providencia stuari. These three enzymes present the restriction sites on the DNA represented below and cut as follows:

EcoRI: restriction site 5 '-XXXXXGAATTCXXXX-3'

3 '-XXXXXCTTAAGXXXXX-5'

cut freeing 5 '-XXXXXG AATTCXXXXX-3'

3 '-XXXXXCTTAA GXXXXX-5'

HaellI: restriction site 5 '-XXXXXGGCCXXXXX-3' 3 '-XXXXXCCGGXXXXX-5'

cut freeing 5 '-XXXXXGG CCXXXXX-3' 3 '-XXXXXCC GGXXXXX-5'

PstI: restriction site 5 '-XXXXXCTGCAGXXXXX-3' 3 '-XXXXXGACGTCXXXXX-5'

cut freeing 5 '-XXXXXCTGCA GXXXXX-3' 3 '-XXXXXG ACGTCXXXXX-5'

In these examples, the X represent the nitrogen bases constituting the DNA contiguous to the restriction site and which do not intervene in the restriction site. In the present, these nitrogenous bases constitute the internal sequences specific to each DNA fragment obtained after digestion with the enzyme. This example shows that the restriction enzyme Hae III cuts DNA with blunt ends, and that the enzymes EcoRI or PstI cut DNA with cohesive ends. In addition to the aforementioned enzymes, EcoR I, Aat II, Sno I, Bgl II, Bln I, Bse AI, Cla I.

According to the present invention, the choice of the restriction enzyme used will depend on the desired number of fragments generated from the DNA of the organism whose imprint is to be made. Indeed, the number of fragments generated by digestion with the restriction enzyme should not be too high in order not to saturate the chip of the present invention after amplification.

The inventors have determined that the number of amplified fragments / number of probes or studs of the chip is advantageously between 0.2. and 0.05, preferably between 0.5. and 0.01. The number of fragments generated by digestion of the DNA of an organism with a restriction enzyme can be easily determined by the AFLP method [11].

When a single restriction enzyme is used, the restriction enzymes generating between 10 ⁶ and 10 ² restriction fragments, preferably between 10 ⁴ and 10 ³ restriction fragments of amplifiable size, preferably from 500 to 1000 base pairs , will be particularly preferred according to the present invention. According to the invention, the choice of the restriction enzyme used can also be a function of the amount of nitrogenous bases constituting the rest of the restriction site sequence after cleavage by the enzyme. In fact, as explained above, it is preferable that the rest of the restriction site sequence is of sufficient length to obtain a specific and stable hybridization between the rest of the restriction site and its complementary sequence on each probe. The rest of the sequence may however be supplemented with an adapter oligonucleotide as set out below.

The restriction enzyme can also be chosen according to the type of cut, frank or cohesive, which it generates by cutting the DNA. A cohesive cut is preferred when an adapter is linked to the fragments as set out below.

The enzymatic digestion step can be carried out by conventional enzymatic digestion methods using restriction enzymes. For example, the work by Sambrook J, Firtsch EF, Maniatis T., 1989 Molecular cloning: a laboratory manual 2 ^nd edition, New-York: Cold Spring Harbor Laboratory Press, cold Spring Harbor, New-YorK describes methods that can be used in the method of the present invention. In general, the conditions indicated by the manufacturer supplying the restriction enzyme are used.

In addition to the reasons explained above, the choice of restriction enzyme used can also be a function of its availability on the market, its cost, the conditions of its use and the protocol for preparing the collection of fragments, etc. The method of the present invention may further comprise a step of ligation of an adapter oligonucleotide, also called, in the present invention, adapter. It can be useful in particular when the rest of the restriction site sequence of the restriction enzyme used is too short to allow stable hybridization with its complementary sequence on the biochip. It can also be useful for the amplification step of the fragments by the polymerization chain reaction (PCR) because it makes it possible to make a primer of sufficient size and complementary to this adapter and to the rest of the restriction site sequence for the action of the polymerase in the process of the invention. Finally, it can be useful for reducing the number of hybridizable fragments on the chip as described below.

This adapter can be prepared by conventional techniques for the synthesis of double-stranded oligonucleotides, for example by synthesis of the two DNA strands complementary to the cohesive ends of a fragment obtained by a specific restriction enzyme, and by hybridization of the strands synthesized in adequate hybridization conditions. The adapters are prepared, for example, by adding equimolar amounts of each strand, heating to 65 ° C, and allowing to cool slowly for about% hour to 20 ° C.

The main thing is that this adapter is of known sequence in order to be able to have its complementary sequence on the probes of the chip. Preferably, it attaches specifically to the ends of the DNA fragments from the restriction enzyme digestion step.

Also, according to a preferred embodiment of the present invention, the restriction enzyme is a restriction enzyme releasing cohesive ends, and the adapter is an oligonucleotide complementary to said cohesive ends. Thus, the adapter attaches specifically to the cohesive ends. This embodiment makes it possible to select with the chip of the present invention the fragments comprising these cohesive cleavages resulting from the action of the determined restriction enzyme and the adapter, and therefore to reduce the number of fragments which can be hybridized on the chip. For example, when the restriction enzyme used is EcoRI, the adapter oligonucleotide can advantageously consist of the following double-stranded oligonucleotide:

5 '-CTCGTAGACTGCGTACC-3' 3 '-CATCTGACGCATGGTTAA-5'.

Other adapters which can be used are for example described for the restriction enzymes PstI and Mspl in document [13]. If another restriction enzyme is used, in place of EcoRI, with other cohesive ends, then the adapter should be modified so as to allow ligation with the cohesive ends released by this other enzyme. According to the present invention, the ligation of the adapter obtained can be carried out by conventional ligation techniques, for example described by Sambrook, Fritsch and Maniatis in Molecular cloning, A laboratory manual, Second edition. It is for example carried out by a ligase, under the sine qua non conditions for the activity of this enzyme.

According to the present invention, the digestion and ligation steps can be carried out simultaneously when the attachment of the adapter to the ends of the fragments does not reconstitute the restriction site. This is for example the case for EcoRI, for example with the adapter presented above. On the other hand, it is not annoying that the restriction site is reconstituted with the adapter when the digestion and the ligation are carried out in successive separate stages.

According to a particular embodiment of the present invention, several restriction enzymes can be used. This can be the case, for example, when the number of fragments obtained with a single restriction enzyme is too large and would lead to saturation of the chip of the present invention.

According to this particular embodiment of the present invention, advantageously, a first restriction enzyme is chosen as a function of its restriction site so that the sequences of the residues of its restriction site are sufficiently long for the reasons explained above. above and in such a way that the DNA fragments obtained with this enzyme are at cohesive ends in order to specifically attach an oligonucleotide thereto adapter determined. A second restriction enzyme is then used, before, during or after digestion with the first restriction enzyme, the ends of the fragments obtained with this second enzyme being incompatible with the determined adapter oligonucleotide.

This embodiment makes it possible to select, on the chip of the present invention, the fragments comprising the cohesive cleavages resulting from the action of the first restriction enzyme thanks to the specific adapter of these cleavages. In fact, only the fragments linked to said specific adapter are amplified during the amplification step, the primer being complementary to the adapter. This therefore constitutes a means of reducing the number of fragments during the manufacture of the genetic fingerprint.

For example, according to the present invention, Taql and EcoRI can be used together to digest the DNA extracted from the organism. Indeed EcoRI generates 40,000 fragments of less than 500 base pairs in mammals. This number can be reduced very significantly by additional digestion of the DNA extracted, for example with a restriction enzyme recognizing four base pairs such as Taql, Mspl, Maell, AluI. The aforementioned adapter for EcoRI can be used in this example.

Amplification can be carried out by any of the chain polymerization methods, called "PCR" techniques. PCR techniques are for example described in Innis MA, Gelfand DH, Sninsky JJ, White TJ, 1990 - PCR Protocols, San Diego, California Academy Press, Inc.

For example, the amplification can be carried out with a single primer whose sequence corresponds to that of the adapter and the restriction site of the enzyme.

Optionally, according to the present invention, one or two additional bases on the 3 ′ side of the primer can be added, this in order to amplify only the fragments comprising the base complementary to this added base. .

In fact, it is important that the chip is not saturated by the amplified fragments so that it can be read and that the manufactured imprint is reproducible. Advantageously, the ratio of the number of amplified fragments / number of probes on the chip is between 0.2. and 0.05, preferably between 0.5. and 0.01.

The addition of one or two nitrogenous bases to the primer is one of the means making it possible to limit, in the process of the present invention, the number of fragments to be hybridized. For example, if the nitrogen base A (or the nitrogen bases AA) is (are) added (s) 3 'to the primer, only the fragments comprising the base T (the bases TT) complementary (s), to the base A (at bases AA) of the 3 'end of the primer, will be amplified. The addition of a single base makes it possible to reduce the number of amplified fragments by four, and thus in certain cases to avoid saturation of the chip according to the present invention. For example, if the restriction enzyme used is EcoRI, then the sequence of the primer may be as follows, possibly with an additional base A, T, C or G on the 3 'side: 5' -GACTGCGTACCAATTCG-3 '.

The amplified fragments are double stranded. They can be labeled during or after their amplification with a fluorescent molecule, either directly during the amplification by the use of a labeled primer. The techniques that can be used are those conventionally used for labeling intended to demonstrate hybridization on a biochip. For example, it can be carried out with Cy3 or Cy5, or by incorporation of a fluorochrome after the amplification reaction. In the latter case, the amplification product is then purified on a column

(QIAquick PCR columns (registered trademark) (QIAGEN) so as to remove the remaining nucleotides and primers before labeling.

The next step consists in denaturing the amplified fragments marked double-stranded so as to transform them into fragments marked single-stranded. This step allows subsequent hybridization of the fragments on the biochip. It can be carried out by any method known to a person skilled in the art which does not alter the fragments and their labeling. A simple heating is sufficient, for example at a temperature ranging from 85 to 105 ° C. Once denatured, the fragments are put to hybridize on a chip according to the present invention, comprising probes adapted to the operating conditions of the abovementioned process. Thus, the probes comprise a constant part recognizing the rest of the restriction site of the restriction enzyme used and, where appropriate, the adapter, and a sequence which varies from one probe to another in such a way that the array of probes recognize the fragments of the collection.

Hybridization is carried out under suitable hybridization conditions. These conditions are easily determined by a person skilled in the art. It will take place in particular at a hybridization temperature which optimizes the specific hybridization, that is to say a temperature making it possible to eliminate the aspecific hybridizations, while favoring specific hybridizations. In general, the melting temperature will be from 45 to 55 ° C, for example at 50 ° C for a probe of about 20 base pairs. The hybridization temperature can be adjusted as a function of the melting temperatures of the oligonucleotides present on the chip of the present invention.

The chip is then rinsed by one of the conventional methods of rinsing the DNA chips after hybridization, for example at room temperature for five minutes with 1 × SSC buffer (containing 0.1% SDS), 2 minutes with buffer 0, 2 x SSC, and finally twenty seconds with 0.02 x SSC buffer. The purpose of this step is to eliminate the non-hybrid fragments and other residues which could hinder the reading of the fingerprint.

After drying, the chip is ready for reading. According to the invention, the reading of the chip can be carried out with any scanner suitable for this type of experiment. For example, the Affymetrix 418 (registered trademark) scanner is suitable for reading.

According to an alternative embodiment of the method of the present invention, it is possible to perform competitive hybridization between the fragments resulting from the amplification step, that is to say comprising the internal sequence proper to each fragment, and synthetic “fixed” fragments which are complementary only to the constant part of the probes fixed on the chip, for example fragments of sequence 5 ′ -GTCATCTACCAATTC-3 ′ in the example of EcoRI and of the abovementioned EcoRI adapter. The fixed fragments can be labeled with a fluorochrome different from that of the fragments resulting from the amplification, for example Cy3 or Cy5 (registered trademark), from the company Pharmacia. The competitive hybridization of the fixed fragments hybridizing on the constant part of all the probes and of the amplified fragments derived from the DNA of the organism (fragments with their own internal sequences) makes it possible to take account of the relative hybridization. of these two types of fragments, and not the absolute value of the fluorescence of the amplified fragments derived from the DNA of the organism. This makes it possible to overcome variations in the quantity of the fragments deposited on the chip. In the case of this variant, the probes fixed on the chip are extended on the side 5 ′, with sequences making it possible to avoid hybridization of the probes resulting from the amplification.

It is of course necessary to adjust the relative quantities of the two types of probe, so as to obtain a suitable signal.

According to the method of the present invention, it is possible to reuse a chip of the present invention on which an imprint has been formed according to the invention, this after treatment and washing of the chip to remove the hybrid fragments therefrom.

The present invention also relates to a genetic fingerprint obtained by a method according to the present invention. This genetic fingerprint can be obtained from the DNA of an organism chosen from an animal, a plant, an insect or a microorganism.

For example, the array of ordered probes of the chip of the present invention being standardized for example to be universal, or to be specific for a genetic marker, for example a genetically detectable character, sex, race, species, variety, etc chosen, the imprint formed according to the process of the present can serve as a control or standard for detecting or not in other organisms or derived products the DNA having served to form the imprint, the genetic marker or the genetically detectable, sex, race, species, or variety.

Also, the present invention also provides a method for identifying an organism comprising DNA or a sample comprising DNA, said method comprising the following steps: a) the manufacture of a first genetic fingerprint of a reference organism by a protocol according to a first method of the present invention, said first fingerprint constituting the genetic imprint of said reference organism associated with the protocol used, -b) the manufacture of '' a second genetic fingerprint from an organism to be identified or a sample to be identified according to the same first process as in step a), and

-c) the comparison of the first imprint obtained in step a) with the second imprint obtained in step b), an identity of the first and of the second imprints revealing if necessary that the organism, or the sample to be identified, has the same identity as, or comes from, the reference organization.

So that two fingerprints of the present invention can be compared, of course, the same method of manufacturing these fingerprints must be used, that is to say the same concentrations of DNA, the same restriction enzyme (s), the same adapter if applicable, and the same hybridization conditions.

The fingerprints can be compared directly ^• or photographed and then compared.

The reference organism may be an animal, and the organism or the sample to be identified an animal or a sample of animal to be identified.

For example, the reference organism may be an animal intended to produce meat for human food, and the sample to identify a piece of meat for sale.

According to the present invention, the reference organism can be a plant, and the organism or the sample to be identified a plant or a plant sample to be identified.

For example, the reference individual may be a reference grape variety, and the sample to identify a sample of grape. According to the present invention, the reference individual can be a suspect, and the sample to identify a human tissue or sample, for example taken at the scene of a crime, likely to come from said suspect.

The present invention also relates to the use of a chip according to the present invention, in a method of identifying an organism or a product derived from this organism, of sex, of race or of variety or of the species of said organism from a sample of its DNA, for example for the purpose of individual traceability of animals or plants and products derived from them.

It also relates to the use of a chip according to the invention for detecting in a microorganism.

The invention brings on the one hand a simplification of the operating protocols compared to the techniques of the prior art because it allows for example in a single operation to obtain a genetic fingerprint of an individual, without any prior development for any species, animal or plant, and on the other hand, it brings a consequent increase in the number of genetic markers analyzed for a lower experimental effort. It is possible thanks to the present invention to analyze several hundred polymorphic markers in a single experiment. It is interesting to note that the fact of being able to avoid migrations on gel and to work by hybridization on a chip allows a further automation of the process, and therefore an industrialization.

In addition, the fact of using a universal chip considerably reduces costs and development times. This universal approach not only makes it possible to carry out genetic fingerprints, i.e. to carry out individual identifications, but also to identify the species, race and sex from one or more reference fingerprints, and this regardless of the organism from which the DNA tested comes.

Other characteristics and advantages of the present invention will appear to a person skilled in the art on reading the examples which follow illustrating the invention, without limitation, with reference to the appended figures.

BRIEF DESCRIPTION OF THE FIGURE

- Figure 1 is a schematic representation of an embodiment of the method of the present invention. EXAMPLES

EXAMPLE 1 Extraction of the Genomic DNA of an Individual

Several samples are taken from different organisms.

DNA can be extracted by the method

Phenol / Chloroform (Sambrook et al. 1989) or using a commercial extraction kit (for example QIA amp

Tissue Extraction Kit (trademark) (Qiageu Company).

Several DNA extracts from different organisms are obtained.

The following examples are made on these different extracts. Figure 1 is a schematic representation of an embodiment of the method of the present invention. In this figure, the extracted sample includes the reference 1.

Example 2: Digestion / Ligation

The restriction enzyme used in this example is EcoRI, and the digestion is carried out at the same time as a ligation with an adapter. These process steps are referenced i) and ii) in FIG. 1, i) representing the digestion of DNA and ii) ligation of an adapter.

The conditions are as follows: 5.5 microliters of DNA (10 mg / μl) extracted by the method described in Example 1, or approximately 55 nanograms of genomic DNA, are added to 5.5 microliters of a compound mixture 100 mM Tris-HCl at pH 7.8, 20 mM of MgCl2, 20 mM of dithiothreitol, 2 mM ATP, 50 μg / ml of BSA (“Bovine Serum Albumin”), 100 mM NaCl, 5 units of EcoRI, 1 unit of T4-ligase, and 1.8 μM of an EcoRI adapter. The whole is carefully mixed. The DNA fragments resulting from the enzymatic digestion are referenced 3 in FIG. 1.

The EcoRI adapter, referenced 5a, 5b in FIG. 1, consists of the following two oligonucleotides: 5 * -CTCGTAGACTGCGTACC-3 • and 5'- AATTGGTACGCAGTCTAC-3 '. The mixture of these two oligonucleotides after heating to 60 ° C followed by slow cooling to room temperature (not shown in Figure 1) gives the adapter which is a fragment of double stranded DNA whose end corresponds perfectly to the cohesive end of EcoRI.

The above mixture is incubated for 2 hours at 37 ° C, then diluted 10 times. The fragments comprising the adapter are referenced 7 in FIG. 1.

Example 3: Amplification

The amplification step, referenced iii) and iv) in FIG. 1, is carried out with a single primer, referenced 9 in FIG. 1, the sequence of which corresponds to that of the adapter and to the restriction site of the enzyme which are used in Example 2, with an additional base on the 3 'side: 5' GACTGCGTACCAATTCG-3 '.

Three microliters of the digestion / ligation from Example 2 are diluted 10 times and added to 22 μL of a mixture making it possible to obtain the concentrations following endings: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.12 mM of each of the dNTPs, 0.2 μM of the EcoRI primer (see sequence above) , 0.5 units of Amplitaq Polymerase (trademark) (Perkin-Elmer).

The chain amplification reaction, referenced iv) in FIG. 1, is composed of the following profile: 2 minutes of incubation at 72 ° C, followed by 30 cycles including denaturation at 94 ° C for 30 seconds, hybridization to 56 ° C for 30 seconds, and an extension of 2 minutes at 72 ° C; Finally, the solution is incubated at 72 ° C for 10 minutes to complete the elongation of all the fragments.

The amplified fragments are referenced 11 in Figure 1. One of the fragments is shown enlarged in this figure in the conventional 3 '5' direction. Appear in order, the adapter 5b, the rest of the restriction site 13, and the internal sequence 13 specific to each fragment.

Example 4: Markings

Part of the amplification products, 5 μL, are labeled with a fluorescent molecule directly during the amplification described in Example 3 above by the use of a primer labeled with Cy3 (trademark), Pharmacia using the Deca-random-prime DNA labeling kit (trademark) from Fermentas.

Another part of the amplification products, 5 μL, are labeled by incorporation of fluorochrome after the amplification reaction. In this last case , the amplification product is purified on a column (QIAquick PCR columns (trademark) from the company QIAGEN, so as to remove the remaining nucleotides and primers before labeling. The results of these labeling are conclusive in both cases.

Example 5: Denaturation and hybridization

Hybridization is carried out on a universal chip of the present invention comprising all the combinations of the variable part for a sequence of 6 nitrogenous bases chosen from A, T, C and G. This chip is referenced in FIG. 1. The reference 22 indicates the sequence complementary to the adapter, the reference 24 indicates the sequence complementary to the restriction site, and the reference 26 the variable sequence. References 22 and 24 therefore indicate the constant part from one probe to another. The sequences of the attached probes correspond to a part of the adapter described in example 2, to the unmodified part of the restriction site, and to a variable part of 6 nucleotides.

Five microliters of the amplified DNA fragments labeled with Cy3 are mixed with 2 microliters of herring sperm DNA (Sigma company) at 20 μg / μL dissolved in an ExpressHyb hybridization solution (registered trademark) (Clontech), and denatured at 96 ° C for three minutes. These labeled denatured fragments are mixed with 15 microliters of the hybridization solution, and deposited on the DNA chip with a device to avoid evaporation.

Hybridization is carried out at 50 ° C overnight.

After hybridization, the DNA chips are rinsed at room temperature for five minutes with 1 x SSC buffer containing 0.1% SDS, 2 minutes with 0.2 x SSC buffer, and finally twenty seconds with 0.02 buffer. x SSC.

After complete drying, the chips and the imprint they contain are ready for reading.

In FIG. 1, the reference 28 indicates a probe hybridized by a fragment.

Example 6: Reading Reading of the chips is carried out with the Affymetrix 418 scanner (trademark).

The fingerprints obtained are legible and reproducible. They indeed make it possible to determine whether two fingerprints obtained from two DNAs come from identical or different organisms. In addition, they make it possible to obtain two identical fingerprints from two different samples from the same organism.

Example 7: Preparation of a support for manufacturing the chip of the present invention.

The substrate used is made of silicon.

A precursor bonding layer for the probe oligonucleotides is deposited on the substrate. It consists of a refractory metal oxide chosen from Ti0 ₂ , Zr0 ₂ , Hf0 ₂ or Ta ₂ 0 ₅ .

The deposited layer is a thin layer with a thickness of between a few nm and 1 μm. This layer is deposited by vacuum evaporation by electron guns at a temperature between about 50 and 200 ° C.

In other tests, it was deposited by ion beam sputtering (IBS), by radio frequency sputtering or magnetron. Vapor deposition of atomic layers (ALCVD) or sol-gel deposition can also be used.

The materials which can constitute the thin layer according to the invention can be deposited, by PVD evaporation techniques (electron guns, IBS, sputtering, etc.), indifferently on glass, plastic, silicon.

The oxides of these metals are very stable with respect to basic solutions, which allows a slow and uniform transformation of the surface.

Without any surface treatment, these materials are hydrophobic. In the case of hafnium oxide, the measurement of the angle of a drop of water leads to a value of

60 °. Depending on the basic treatment performed, the drop angle varies from 35 ° to 6 °.

Supports thus treated were used to grow oligonucleotides by synthesis in situ on a support consisting of a layer of Hf0 ₂ on a silicon substrate. For a sample of low hydrophilicity (35 °), the fluorescence signal, after hybridization of probes of 20 mothers by complementary targets, is weak and not uniform. A highly hydrophilic sample (6 °) shows a clear improvement, mainly in terms of uniformity. The uniformity obtained was compared with that of a solid support commonly used, namely a support formed of a silicon substrate covered with a layer of thermal oxide 0.5 μm thick. Compared to this solid support of the known art, the support according to the invention (Hf0 ₂ on Si) provides an improvement in the uniformity of the fluorescence signal.

The same result was obtained with a glass substrate covered with a layer of Hf0 ₂ after immobilization by covalent bonding of probes from 20 pre-synthesized mothers.

This type of deposit can also be used for biochips using the method of immobilization of probes by electrostatic interactions.

The deposition of these refractory metal oxides allows all types of grafting used in biochip technologies, namely any support (glass, silicon, plastic) and any type of biological grafting technique (in situ synthesis, immobilization by covalent bond or electrostatic connections).

The results of the measurements show an improvement in the uniformity of the fluorescence signals compared to the state of the art.

This technique makes it possible to manufacture the probe arrays of the present invention, and therefore a universal chip according to the invention. REFERENCE LIST Mini-satellite probes

[1] JEFFREYS AJ, WILSON V, THEIN SL (1985a) Hypervariable "minisatellite" regions in human DNA, Nature, 314, 67-73.

[2] JEFFREYS AJ, WILSOΝ V, THEIΝ SL (1985b) Individual-specifies "fingerprints" of human DΝA, Nature, 316, 76-79.

RAPD

[3] WILLIAMS JGK, KUBELIC AR, LIVAK KJ, RAFALSKI

JA, TIΝGEY SV (1990), DΝA polymorphisme amplified by arbitrary primers are useful as genetic markers,

Nucleic Acids Research, 18, 6531-6535. [4] WELSH J, MCCLELLAND M (1990) Fingerprinting genomes using PCR with arbitrary primers, Nucleic Acids

Research, 18, 7213-7218.

[5] WELSH J, PETERSEN C, McCLELLAND M (1991)

Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping, Nucleic Acids Research, 19, 303-306. [6] WELSH J, McCLELLAND M (1991) Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers, Nucleic Acids Research, 19, 5275-5279.

[7] MUNTHALI M, FORD-LLOYD BV, NEWBURY HJ (1992),

The random amplification of polymorphic DNA for fingerprinting plants, PCR Methods and Applications, 1,

274-276. [8] MICHELI MR, BOVA R, CALISSANO P, D'AMBROSIO E

(1993) Randomly amplified polymorphic DNA f ingerprinting using combinations of oligonucleotide primers, BioTechniques, 15, 388 - 390.

ISSR [9] ZIETKIEWICZ E, RAFALSKI A, LABUDA D (1994), Genome fingerprinting by simple sequence repeat (SSR) - anchored polymerase chain reaction amplification, Genomics, 20, 176-183.

[10] KANETY R, CHENG X, BENNETZEN J, BRENT E (1995), Assessment of genetic diversity in dent and popcorn (Zea mays L.) inbred lines using inter-simple sequence repeat (ISSR) amplification, Molecular

Breeding, 1, 365-373.

AFLP

[11] VOS P, HOGERS R, BLEEKER M, REIJIANS M, VAN DE LEE T, HORNES M, FRIJTERS A, POT J, PELEMAN J, KUIPER M, ZABEAU M (1995) AFLP: a new technique for DNA fingerprinting, Nucleic Acids Research, 23, 4407-4414.

[12] AJMONE -MARSAN P, VALENTINI A, CASSANDRO M, VECCHIOTTI -ANTALDI G, BERTONI G, KUIPER MTR (1998), AFLP ™ markers for DNA f ingerprinting in cattle, Animal Genetics, 28, 418 -426.

DArTs

[13] JACCOUD D, PENG K, FEINSTEIN D, KILIAN A (2001), Diversity arrays: a solid state technology for sequence information independent genotyping, Nucleic Acids Research, 29, e25.

Claims

1. Hybridization chip for a collection of single-stranded fragments of a deoxyribonucleic acid originating from an organism, said collection of fragments being obtained in particular by digestion of said deoxyribonucleic acid with at least one restriction enzyme having a determined restriction site, each fragment comprising a sequence residue from said restriction site and, adjacent to said sequence residue, an internal sequence specific to each fragment; said chip comprising a support on which is fixed an ordered array of nucleic acid probes, said probes comprising a first and a second part of sequence, the first part of sequence being constant from one probe to another and at least complementary to the rest of the sequence of the determined restriction site, and the second part of the sequence, contiguous to said first part of the sequence, being variable from one probe to another and consisting of determined combinations of nitrogen bases chosen in such a way that the ordered array of probes includes the probes capable of hybridizing specifically to the different nucleic acid fragments of said collection.

2. Hybridization chip according to claim 1, in which the first part of the sequence which is constant from one probe to another of the chip and complementary to the rest of the restriction site sequence of the restriction enzyme used is in addition to a sequence-specific adapter oligonucleotide, said collection of single-stranded fragments being obtained in particular after ligation of a sequence-specific adapter oligonucleotide at the ends of the fragments of said organism deoxyribonucleic acid obtained by digestion with the restriction enzyme determined.

3. Hybridization chip according to claim 1, in which said first part of sequence is complementary to the rest of the restriction site of the restriction enzyme used for example chosen from EcoRI, Aatll, Snol, Bgl II, Bln I, Bse AI, Cla I.

4. The chip of claim 1 ^" or 2, wherein said first portion of sequence is complementary to the rest of the restriction site of the restriction enzyme EcoRI.

5. Chip according to claim 1 or 2, in which each second part of sequence variable from one probe to another has a constant length chosen from 4 to 12 nitrogen bases.

6. Chip according to claim 1 or 2, in which each second part of sequence variable from one probe to another has a constant length chosen from 4 to 10 nitrogen bases.

7. The chip of claim 1 or 2, wherein each second portion of variable sequence from one probe to another has a constant length chosen from 4 to 12 nitrogenous bases chosen from adenine, cytosine, guanine and thymine.

8. A chip according to claim 1 or 2, in which each second part of sequence variable from one probe to another has a length of 6 nitrogenous bases chosen from adenine, cytosine, guanine and thymine.

9. Chip according to claim 1 or 2, in which each second part of variable sequence from one probe to another has a length of 6 nitrogen bases and consists of determined combinations of nitrogen bases chosen from adenine, cytosine , guanine and thymine, so that the array of probes includes 4 to ⁶ different possible probes.

10. The chip according to claim 4 when it depends on claim 2, in which the adapter oligonucleotide has a sequence complementary to one or to one of one of the following two oligonucleotides: 5 '-CTCGTAGACTGCGTACC-3'; and

5 '-AATTGGTACGCAGTCTAC-3'.

11. A method of manufacturing a genetic fingerprint of an organism comprising the following steps: extraction of double-stranded DNA from said organism, determination of the concentration of double-stranded DNA extracted and if necessary dilution of the extracted DNA to a concentration of 1 to 20 ng / 1, digestion of the extracted DNA before or after dilution with means of at least one restriction enzyme having a restriction site determined so as to obtain double-stranded fragments comprising a sequence residue of said restriction site, and, adjacent to said sequence residue, an internal sequence specific to each fragment; optionally ligation of a double-stranded adapter oligonucleotide with a determined sequence at the ends of the double-stranded fragments obtained by digestion of said DNA to obtain suitable double-stranded fragments, amplification of the double-stranded fragments, optionally linked to the double-stranded adapter oligonucleotide, by a polymerase chain reaction by means of a primer complementary to said remainder of the sequence of said restriction site and optionally to the double-stranded adapter oligonucleotide, labeling of the double-stranded fragments, optionally linked to the double-stranded adapter oligonucleotide, during or after their amplification, by means of a marker,

Denaturation of the marked double-stranded fragments, possibly linked to the double-stranded adapter oligonucleotide, so as to obtain a collection of single-stranded fragments of the acid deoxyribonucleic coming from the organism possibly linked to the single-stranded adapter oligonucleotide, hybridization of said collection of fragments on a chip according to claim 1 when the fragments are not linked to the single-stranded adapter oligonucleotide, or on a chip according to claim 2 when the fragments are linked to the single-stranded adapter oligonucleotide, - rinsing of the chip in particular to remove the non-hybrid fragments and drying.

12. The method of claim 11, wherein the restriction enzyme used is chosen from EcoR I, Aat II, Sno I, Bgl II, Bln I, Bse AI, Cla I.

13. The method of claim 11 comprising the step of ligating a double-stranded adapter oligonucleotide, in which the restriction enzyme used cuts the DNA leaving cohesive ends, the adapter oligonucleotide chosen having an end which corresponds perfectly to the cohesive end of the restriction enzyme used.

14. The method of claim 13, wherein the restriction enzyme used is EcoRI.

15. The method of claim 13, wherein the adapter oligonucleotide consists of

The following double-stranded oligonucleotide: 5 '-CTCGTAGACTGCGTACC-3'

3 '-CATCTGACGCATGGTTAA-5'.

16. The method of claim 13, wherein the digestion and ligation steps are carried out simultaneously.

17. The method of claim 11, wherein two restriction enzymes are used to digest the extracted DNA.

18. The method of claim 17, wherein the enzymes are Taql and EcoRI.

19. The method of claim 17 or 18 comprising the step of ligating a double-stranded adapter oligonucleotide, in which the two restriction enzymes used cut the DNA leaving cohesive ends, the adapter oligonucleotide chosen having an end which corresponds perfectly at the cohesive end of only one of the two restriction enzymes used.

20. Genetic fingerprint obtained by a process according to any one of claims 11 to 19.

21. A genetic fingerprint according to claim 20, in which the organism is chosen from an animal, a plant, an insect or a microorganism.

22. Method for identifying an organism comprising DNA or a sample comprising DNA, said method comprising the following steps:

-a) the manufacture of a first genetic fingerprint of a reference organism by a protocol according to the method according to claim 11, said first fingerprint constituting the genetic fingerprint of said reference organism associated with the protocol used, -b) the manufacture of a second genetic fingerprint from an organism to be identified or from a sample to be identified according to the same protocol as in step a), and

23. The method of claim 22, wherein the reference organism is an animal, and wherein the organism or sample to be identified is an animal or a sample of animal to be identified.

24. The method of claim 22, wherein the reference organism is an animal intended to produce meat for human consumption, and the sample to be identified is a piece of meat intended for sale.

25. The method of claim 22, wherein the reference organism is a plant, and wherein the organism or sample to be identified is a plant or a sample of plant to identify.

26. The method of claim 22, wherein the reference individual is a reference grape variety, and wherein the sample to be identified is a sample of grapes or wine.

27. The method of claim 22, wherein the reference individual is a suspect, and the sample to be identified is a human tissue likely to come from said suspect.

28. Use of a chip according to claim 1 or 2, in a method of identifying an organism or a product derived from this organism, of sex, of race or of variety or species of said organism. organism from a sample of its DNA.

29. Use of a chip according to claim 1 or 2 in the identification of genes involved in detectable characters such as diseases of genetic origin.

30. Use of a chip according to claim 1 or 2 for the purposes of individual traceability of animals or plants and products derived from them.

31. Use of a chip according to claim or 2 for the purpose of detecting a microorganism.