WO2010070462A1

WO2010070462A1 - Microarray for the identification of the presence or absence of gmo in samples comprehensive of vegetable material

Info

Publication number: WO2010070462A1
Application number: PCT/IB2009/052962
Authority: WO
Inventors: Cinzia Colombi; Carlo Massimo Pozzi
Original assignee: Fondazione Parco Tecnologico Padano
Priority date: 2008-12-17
Filing date: 2009-07-08
Publication date: 2010-06-24
Also published as: IT1392539B1; ITRM20080673A1

Abstract

The present description discloses a microarray capable of simultaneously identify the presence or the absence, in a sample, of each of 135 transgenic events that allowing the identification of genetically modified organisms (GMO), a kit for the detection of GMO, a method for the contemporaneously identification, in a sample, of the presence or absence of, from at least 15 to at least 135, different transgenic events and/or materials coming from one or more GMO.

Description

MICROARRAY FOR THE IDENTIFICATION OF THE PRESENCE OR ABSENCE OF GMO IN SAMPLES COMPREHENSIVE OF VEGETABLE

MATERIAL DESCRIPTION The present description discloses a microarray able to simultaneously identify, in a sample comprising plant material, the presence or absence of each of at least 15 to at least 135 transgenic events that allow of identify genetically modified organisms (GMO), a kit for the detection of GMO, a method for the simultaneous identification, in a sample, of the presence or absence of, from 15 to 135 different transgenic events and/or of material deriving from one or more GMO.

STATE OF THE ART

DNA microarrays, also known as DNA chip or gene chip, are a important tool for the so called "nanotechnologies".

On the market since 1996, microarrays allow the simultaneous analysis of the activity of dozen of thousands of genes. The chips are made with a plurality of DNA molecules (called probes) laid on a known position on a support to form a micro grid (from which the name microarray) that allows to identify each of said probes in an unambiguous way. The support is, usually, a microscope slide. Every probe is constituted of a single helix DNA segment of interest. The microarray slide is a very powerful diagnostic tool capable to identify, in a single experiment, the presence/absence of a high number of targets. The chips take advantage of an important DNA property, which is the complementary bases match (the T matches with the A and the G with the C) in its structure. The diagnostic technique consists in the presence of a luminous signal (emitted by a fluorophore at different wave lengths) in correspondence to the hybridization between the target fragment labelled with the fluorophore and the corresponding probe bound to the microarray slide. This binding, with subsequent light emission, shows that in the group of analysed probes a DNA fragment complementary to the probe that is "lighted" is present and, consequently, allows to know the identity of the target fragment. There are several techniques for labelling the DNA (or cDNA) analysed (direct, indirect) through the creation of link with fluorophores emitting emit light at different wave lengths (usually in the red and in the green). The probe on the microarray slide is not bound to the fluorophore, therefore the microarray slide that is not hybridised, when observed under a confocal scanner does not show any luminous signal. A "limit" in the use of microarray technology for the GM organisms (genetically modified) detection, is represented by the need to perform a PCR (Polymerase Chain Reaction) amplification of the target DNA, prior to the phase of example a gene or one or more gene portions, in the transgenic cassette used for the GMO production) is usually present in a single copy into the transformed genome, which makes extremely difficult the match and the hybridisation between the single copy of the gene and the corresponding probe on the microarray slide. A pre-hybridization amplification reaction allows to increase the copy number of the target gene (or fragments thereof), easing the hybridization event with the complementary probe on the microarray slide.

The PCR amplification reaction usually requires the use of specific primer pairs for the amplification of different regions of the transgenic cassette. Nevertheless, every GMO is different from the others for some characteristics of the gene cassette itself (e.g. different gene of interest).

A tool exists on the market, Eppendorf DualChip GMO Microarray, which implies a method based on the use of the microarray technology. In that method the target sequences are amplified by multiplex PCR, the amplicons are biotinylated and labelled by Silverquant method. Three multiplex PCR reactions are carried out with of specific primer pairs for the detection of 11 different transgenic events (approved by EC), 7 plant species and a negative control for Cauliflower Mosaic Virus (CaMV) contamination. Hence, the "DualChip" tool, show a high limitation due to the need of performing pre-hybridisation amplifications of the microarray slide using a number of specific primer pairs that depends on the number of probes on the slide itself. The DNA of the sample to be analysed must be amplified, by PCR reaction, using primer pairs that are specific for the amplification of fragments capable of hybridising with the probe blocked on the microarray slide. The greater the probes number (and consequently the number of different transgenic events, GMO, to be detected) and the greater the number of specific primer pairs involved in the PCR amplification of target fragments having a sequence complementary to the probes blocked on the microarray slide. Obviously, this protocol involves: a limitation in the number of probes on the microarray slide (and hence of the number of detectable GMO); a high waste of time (used to carry out every PCR reaction with different primer pairs); a high cost due to the reagents used in the amplification phase. In order to minimise the problems linked to the technique and hence to the need of carrying out this number of pre-hybridisation amplifications, it is possible to carry our "Multiplex PCRs" which allow the combination, in a single amplification reaction, more than one pair of specific primers. There is however, a limit to the number of primer combinations that is possible to introduce in a multiplex and this corresponds averagely to 7/9 different primer pairs. The use of multiplex partially reduces the problems linked to the amplification of the target DNA, but it is not sufficient to overcome them. Likewise, the patent application US 20070117106Al titled "Identification and/or quantification method of nucleotide sequence (s) elements specific of genetically modified plants on arrays" describes a method for the identification of transgenic events in the plants genome that foresees an amplification phase of the target sequence by standard PCR and a detection by hybridisation probes blocked on a microarray.

There are several publications in literature (indicated in the bibliography below) describing the use of a method for the identification of foreign genes, viral or GM, in which the target genetic material is digested by suitable restriction enzymes, ligated to adapters allowing the use of a single primer pair for the amplification, and amplified by PCR. Among these, T. R. Allnutt et al. underlines the objective difficulty in the application of the aforesaid method to the plant genome given the wideness of the plant genome that, notoriously, can often be polyploid. The practical problem derives from the fact that, the greater is the analysed genome and the greater is the probability that the primer recognising the adapter amplifies also aspecific sequences. The paper underlines the difficulty found in the detection of 6 transgenic events in plant material.

The most recent work, published by Arkiditis et al in 2008, underlines the of finding suitable means for the identification of GMO events in samples containing plant material (e.g. food) and underlines, once more the difficult identification of this events. One of the main reasons is due to the scarcity of information given on GMO by the producing companies and on the secrecy surrounding the exact preparation processes of these plants.

Consequently, the identification of GMO events in plant material is to date very limited and it is desirable to provide effective means for the identification of said GMOs.

SUMMARY OF THE INVENTION

The following invention finds application, in particular, in the agro-industrial sector answering to the needs of Small and Medium Companies operating in the market of food production, of having at their disposal a method that is fast, reliable and non expensive for the determination of the presence of Genetically Modified Organisms in plant material samples, hi particular, what described in the present description applies to the search of Genetically Modified Organisms (GMO) in products destined to human alimentation or to the preparation of food for high income animals, in consideration of the high percentage of basic elements of plant origin, destinated to a human alimentary use or destinated to animal feed, introduced from Countries that use transgenic coltures. The EC Regulation n. 1829/2003 of the European Parliament and of the Council, relative to genetically modified food and feed, provide that it is not possible to put on sale, use or modifiy, a product according to article 15, paragraph 1 of the aforesaid Regulation (GMO destined to animal feed; feeds containing or constituted of feed; feed produced from GMO), unless that a regular autorization and labele has been released for it (avoidable only when the foods containing material that contains, is constituted or productd from GMOs are present in proportion of not higher than 0.9% of the food components individually taken into account, or of the single component foods, provided that this presence is accidental or technically inevitable - lower than 0.5%). At present the diagnostics linked to the GMO is essentially based on two diagnostic methods accepted from the international scientific community:

PCR (Polymerase Chain Reaction) "qualitative": method of analisys validated from JRC of Ispra (Screening method for the identification of GMO in food: i) detection of the CAMV 35S and NOS terminator by means of PCR); ii) "quantitative" PCR (Polymerase Chain Reaction) analysis carried out by Real - Time PCR

Both this methods, however, require a remarkable investment of materials and time, for their realization, due to the need of using, normally, different specific primers for each transgenic event to be detected (and, consequently, different reactions of amplification by PCR). The diagnostic tool herein described allows an initial screening of samples comprising material of plant origin with a simultaneous detection of the presence or absence of at least 15 different transgenic events up to 135 different transgenic events. On positive samples it will then be possible to concentrate the attention and to proceed with classic diagnostic methods (e.g. Real - Time PCR).

The present description discloses also that a method of amplification and of use of the microarray, generally known but considered as not easily applicable to the plant genome, can be applied to genetic material deriving from every type of plant matrix for different diagnostic and of research scopes. Moreover, the designed probes herein described can be employed also with other diagnostic instruments (qualitative PCR; quantitative PCR Real - Time), securing a high grade of diagnostic capacity.

Unexpectedly, the inventors of the present description have successfully used the digestion method — ligation with adapter sequences — amplification with suitable primers, indicated as problematic on plants in the state of the art, on a large pattern of plant species and have succeeded in the identification of highly specific sequences. Sequences allowing the simultaneous detection of the presence or absence of at least 15 transgenic events up to 135 transgenic events, that are, as described in the state of the art, identifiable in a difficult way for the reasons reported above. With respect to what is available to date, the matter described in the present description allows the simultaneous detection, with a single research tool, of almost twice (15 in comparison with 9) of the transgenic events detectable with current commercial kits (up to 135 transgenic events in comparison with 9).

The present description discloses a microarray capable of simultaneously identify the presence or absence, in a sample, of each of at least 15 different transgenic events up to each of 135 different transgenic events allowing the identification of genetically modified organisms (GMO), a kit for GMO detection, a method for the simultaneous identification, in a sample, of the presence or absence of each of at least 15 different transgenic events up to each of 135 different transgenic events and/or of material deriving from one or more GMO.

The present description discloses, therefore, a microarray comprising, as probes blocked on said microarray, nucleotide sequences comprised in the group from SEQ ID NO 5 to SEQ ID NO 107, selected in order to simultaneously detect the presence or absence of each of at least 15 different transgenic events up to each of 135 different transgenic events in samples comprising plant material; a kit for the detection of the presence or absence of transgenic events and for the their identification in sample comprising plant material said kit comprising, one or more microarrays according to claim 1 and, optionally, one or more aliquots of reagents for the detection of said probes; a kit for the identification of GMO in samples comprising plant material, comprising one or more microarrays according to claim 1, optionally one or more aliquots of reagents for the detection of said probes, optionally a matrix or a computer support containing data enabling the user to identify the transgenic events detected by the microarray and/or GMO depending on the combination of said transgenic events; a method for the identification with a single test, in sample comprising plant material, of the presence or absence of each of at least 15 different transgenic events up to each of every of 135 different transgenic events. DETAILED DESCRIPTION OF THE FIGURES

Figure 1: Southern blotting. The DNA (Standard FLUKA - mon810 5%) was digested with two restriction enzymes (see examples) and loaded on a gel, then transferred on a filter, in six different dilutions (reported in the picture in the sequence 1, 2, 3, 4, 5, 6, ): 1:10, 1:20, 1:50, 1:100, 1:250, 1:500. Figure 2: Southern blotting. The DNA (Standard FLUKA - mon810 5%) was amplified with the kit for the Whole Genome Amplification (GE Healthcare - GenomiPhi V2 DNA Amplification Kit) and transferred, in six different dilutions (1 :10, 1:20, 1:50, 1:100, 1 :250, 1:500), on a hybridisation filter (see examples).

Figure 3: Represents a virtual slide usable for the interpretation of the microarray of the invention, with 88 sequences of interest, 20 sequences of endogenous as positive control, 2 sequences as negative control. Four copies for each gene are indicated.

The black full circles are negative controls, the grey empty circles represent defective points, their presence helps in the slide orientation, and the black empty circles represent the expressed genes.

Fig 4: At the start of the data base GMOGem it is possible to choose between 4 options: File, Registry, Search, and Info. Entering in "Coordinates for sequences preview" (window "Registry") it is possible obtain information on the reciprocity between spot and sequence and, consequently, transgenic event.

Fig. 5: Example of "aimed" research inside the data base, in the case in which a particular transgenic event is expected: the name of the expected event GMO (5345 in our example) is inserted and in the"COORDINATES" window (at the right hand side of the screen) the corresponding sequences (spots) that shall turn on, on the microarray slide will appear (name and coordinate).

Fig. 6: Example of research inside the data base, in the case in which there is no expectation of a particular transgenic event. Upon entry in the "SEARCH" mode a video with three windows appears (on the right hand side of the screen), corresponding to the three co-ordinates necessary for the identification of the sequences corresponding to the spots present on the slide: Block, Row, and Column.

Upon introduction of the co-ordinates corresponding to the spot that is lighted on the microarray slide (is possible to carry out this research operation of search for a spot at the time), in order to obtain information on which is the sequence to which the spots refers and in which event/s the analysed sequence is present.

Fig. 7: Example of research carried out on the basis of the spot co-ordinates, hi this case the spot under examination has co-ordinates: Block 1, Row 2, Column 3, corresponding to the genie sequence Xl 5855 (endogenous gene), present in the transgenic event 5345.

Fig. 8: In the section "Preview of the spots for an event" of the data base it is possible to identify the spots corresponding to each transgenic event. By positioning the cursor on each spot the name of the corresponding sequence appears and upon clicking of the left key of the mouse all the information on the sequence itself are displayed (see Fig. 9).

Fig. 9: Example of reciprocity between spot and sequence. By positioning the cursor on a spot and upon clicking of the left key of the mouse all the information on the sequence corresponding to the spot are displayed.

DETAILED DESCRIPTION OF THE TABLES

Table 1 contains the sequences of the probes of the invention, the gene of derivation (with explanation of its function) and the event that contains it. Table 2 shows a list of the 135 events of the description and of the related organism modified in order to carry out each event.

The table 3 shows a list of 15 events of interest and of the groups of sequences that identify said events.

The table 4 shows combinations that lead to the identification of 135 events with 95 different combinations. As it possible to note in the present table, besides the indication of the sequences identifying each event, also sequences of endogenous genes are reported acting as positive controls of the hybridisation and sequences that are negative controls.

The table 5 shows the correlation between each single sequence and the different transgenic events herein listed.

Every combination indicate the sequences that shall hybridise (and hence the corresponding spots that shall turn on, on the microarray slide), when the transgenic event is the one indicated in the column "Event Name". Each combination contains sequences that can be present also in other combinations, but coupled with different sequences.

The table 6 shows the comparison, in the hybridisation efficiency, between multiplex PCR, using specific primers for every probe blocked on the slide, and Whole Sample Amplification.

- PCR Matrix l_l l-12-07_elab: hybridisation carried out with PCR amplification, of the target DNA, using primers that amplifies in a specific manner the region comprising the spotted probe (a PCR for each probe).

- AFLP Matrix l_24-01-08_elab: hybridisation carried out with the Whole Sample Amplification method (digested genomic DNA, ligated with adapter and amplified twice with adapter primers) and 16 hours of hybridisation at 48⁰C (25% of Formamide).

- AFLP-3h_7-02-08_elab: hybridisation carried out with the Whole Sample Amplification method (digested genomic DNA, ligated with adapters and amplified twice with adapter primers) and 3 hours of hybridisation at 50°C (0% of Formamide).

DETAILED DESCRIPTION OF THE INVENTION The microarray according to the present description comprises, as probes blocked on the slide or on a suitable support, one or more copy of oligonucleotides (probes) having nucleotide sequences comprised in the group from SEQ ID NO 5 to SEQ ID NO 107 in precise positions, in order to form a micro grid, and selected in said group based on the data provided in the tables, for the simultaneous detection of the presence or absence of at least 15 transgenic events indicated in table 2. For each of these events, sequences suitable for the detection and the identification of each event are listed in table 4. As can be seen in the table, for each event a plant control sequence is used, by way of example chloroplast DNA (SEQ ID 56) that, obviously may be substituted with other analogous typical plant sequence without need of particular teachings. Given the detailed information present in table 2 and in the various tables of the description, all the necessary data for the realization of a microarray for the simultaneous detection of the presence or absence of at least 15 transgenic events up to all the 135 the transgenic events herein described are provided.

As the description tables allow locating which probes are needed for the identification of each event, it will be sufficient for the skilled person to develop a microarray comprising the probes needed for the detection of the presence or absence of at least 15 events up to the 135 events. Any combination of events may be carried out from the teachings provided in the present description. The skilled person may hence carry out microarrays for the detection of the presence or absence of fifteen or more of the events herein indicated said 15 events being selected in any combination among the 135 indicated in the tables of the present description.

The selection of the events principally concerns the interests of the user. By way of example, Table 2 identifies fifteen events of sufficiently common interest that can be detected by the microarray of the invention. It is clear that any between the 135 events indicated in the present description may be selected in order to be detected on the microarray of the invention as the sequences suitable for its detection are indicated in the present invention. As each of the 135 events, indifferently, can be selected by a skilled person reading the tables herein reported, and since the information needed for the punctual identification of each of the 135 events is herein supplied, it is evident that the present description provides the support essential to the realization of a microarray for the detection of each of the events indicated in table 2. Nevertheless, being a multiple detection undoubtedly more useful for the research rather than the detection of one or few events at the time, in the present description are provided, sequences that can be detected in a single experiment, namely, the microarray allows the simultaneous detection of the presence or absence of indeed 135 transgenic events. In the present description the sequences suitable to the detection of every of the 135 events are indicated, said sequences can be blocked on a single microarray and can be detected in a single experiment as they are selected so that they do not present any cross-hybridization and in order to have a gc content and a length allowing the same detection protocol for all the sequences 120used.

It is clear that is possible to carry out microarrays allowing the simultaneous detection of one or more of each events listed in table 2, when the events to detect are n, for n greater than one, they can be any combination of n of the 135 events indicated in table 2.

The microarrays of the present description also allow, depending on the detected events, to identify the presence of GMO in the analysed samples and, depending on the combination of the events that are present, to identify particular genetically modified organisms.

Thus, the identification of these events, characteristic of GMO plant organisms, allows the detection of the presence GMO material in the plant sample. These probes will be attached to the support by standard techniques, known to the skilled person, and they are highly specific for the detection of transgenic events reported in table2. Table 5 indicates which probes are linked to each transgenic event, as can be seen in the table, SEQ IDs NOs 15-32 are positive control probes and probes 34 and 107 are negative control probes (Bos taurus). The presence of positive and negative control probes is clearly useful for having a test in which it is possible to estimate the reliability of the results. The skilled person will appreciate that the choice of the control probes can be carried out without need of inventive skills or of particular experimentation when starting from what is exemplified in Table 5. It is therefore clear that the present invention is not limited to the control probes in the table and in the sequence listing and that the presence, on the microarray herein described, of any suitable control probe, feasible for the skilled person, is considered an embodiment of the invention.

The slide of the present invention may hence comprise only probes detecting the transgenic events, selected in the group of SEQ IDs NOs 5-14, 33, 35 - 106 and may also comprise control probes, positive or negative, as reported in table 5. In an embodiment of the invention they may be, respectively, SEQ IDs NOs 15-32 and SEQ IDs NOs 34 and 107.

As already stated, other control probes may be laid on the microarray without use of inventive activity or without the need of experimental burden.

The negative control probes will be probes from organisms that are not under investigation (e.g. organisms belonging to the animal kingdom), the length may still be of about 50 nucleotides and the selected probe will not be cut by the selected restriction enzymes. All this is easily achievable by a skilled person. Moreover, specific negative control probes from organisms used in the vectors for the realization of GMO may be used so to identify any possible contaminations that would lead to false positives in some cases. For example, parts of the CMV are used for the construction of vectors used for the transformation of GMO plants, the use of a specific probe for this organism but absent from these said vectors, may indicate to the researcher the possible presence of viral contaminants that can give false positives. Also in this case the realization of similar probes is easily achievable by the skilled person.

The positive controls can be any probe having the indicated characteristic of length and restriction map, specific for the plant organisms under investigation. Each probe can be anchored on the microarray in one or more copies (in the same site) a greater number of copies clearly have the function of increasing the intensity of the positive signal on the slide. The microarray herein described will allow, according to the results obtained by hybridising the DNA extracted from a sample comprising plant material, to identify the presence/absence of each of the 135 transgenic events reported above.

Moreover, depending on the detected events, the original transgenic plants may be identified even for those GM plants for which transgenic events that are present are known or will be known when such plants comprise transgenic events detectable by the microarray described above. The preparation of said microarray (i.e. the synthesis of the oligonucleotides- probes and the binding thereof on suitable supports, e.g. slide) is known to the person skilled in the art and does not require further indication in the present description. The preparation of microarray may also be requested to companies providing this service. Therefore, no further details on the preparation of the object per se are necessary to carry out what described in the present documentation.

A non restrictive list of plants, whose material is analysable, is herein reported: Potato (Solarium tuberosum), wheat (Triticum aestivum), melon (Cucumis meld), Papaya (Carica papaya), soybean (Glycine max), beetroot (Beta vulgaris), cotton (Gossypium hirsutum), turnip (Brassica napus), maize (Zea mays), lentil (Lens culinaris), tomato (Lycopersicon esculentum), flax (Linum usitatissimum), chicory (Cichorium intybus), pumpkin (Cucurbita maxima), rice (Oryza sativa), sunflower (Helianthus annuus), barley (Hordeum vulgare), Medicago sativa, Alfalfa.

This list, obviously not at all exhaustive, is to give an idea on the system versatility and the wide range of species and plant families that may be analysed by the microarrays herein described.

In one embodiment of the invention allowing a very fast and inexpensive screening, the genetic material to be analysed will be digested with at least one restriction enzyme whose restriction site is absent in SEQ IDs NOs 5-107 (or, if not using SEQ IDs NOs 15-32 and/or 34, 107 as control sequences, said restriction site will also be absent from the selected control sequences ), the cut with a couple of these enzymes allows to obtain a pool of DNA fragments that have, averagely, suitable dimensions for a optimal amplification by PCR.

This fragments thus digested, will be linked using a suitable ligase (e.g. T4 ligase or other ligases known to the skilled person) to "adapter" sequences specific for the protruding regions generated by the enzymes used and designed in order to also bind themselves to a universal primer pair so to allow the amplification, with a single primer pair, of all the fragments obtained.

The adapter sequences as herein defined, therefore, will comprise a region complementary to the sticky end generated by restriction enzyme and a region complementary to a universal primer for the amplification.

The presence, in the adapter sequences, of the region complementary to the universal primer, allows the use of a single of primer pair even when more enzymes are used in order to obtain the fragments. This technique allows an effective amplification and therefore renders the reading of the microarray more efficient as the addition of copies of the genetic material to be analysed increases the probability of hybridisation of the sequences of interest with the corresponding probes bound to the slide. The sequences may be labelled by direct or indirect labelling using any type of technique known to the person skilled in the art.

Commercial fluorophores (e.g. Cy fluorophors family), biotin, digossigenin and other fluorochromes or fluorophores known commonly used in the microarray technique may be used. Due to the rapid evolution of the tools for signal detection, it is evident that the use of any future labelling technique suitable for use on the microarray herein described would be obvious for the skilled person.

In an embodiment, the enzymes EcoRI and/or Msel may be used for the cleavage of the DNA under investigation, and, the sequences SEQ IDs NOs 1 and 2 may be used as adapters for EcoRI, and the sequences SEQ IDs NOs 3 and 4 may be used as adapters for Msel. All these adapter sequences allow the amplification with primers of SEQ IDs NOs 124 and 125.

It is evident that, as the sequences of the protruding end generated by enzymatic cleavage are known, adapter sequences, for use with other universal primers, may be designed without any effort of inventive activity

The same instructions herein provided may be used for other suitable enzymes as described above. Labelling can be carried out during the amplification reaction or the primers for the amplification may be pre-labelled.

The present description also discloses a kit for the identification of transgenic events in samples comprising plant material, comprising one or more microarray as described above and one or more aliquots of reagents for the detection of said probes.

The microarray described above may be used with DNA, preferably amplified with any amplification method, including the direct amplification of DNA complementary to the probes, the kit may comprise 1 or more ready-to-use microarray and the micro grid reading key that allows the identification from each of at least 15 events up to each of 135 transgenic events, based on data reported in the tables. The kit may further contain, one or more of the following materials: one or more aliquots of suitable restriction enzymes and buffers for the use thereof, one or more aliquots of suitable adapter sequences, one or more aliquots of ligation reagents, one or more aliquots of PCR reagents, one or more aliquots of primers suitable for the adapter sequences optionally labelled, one or more aliquots of fluorophores or reagents necessary for the labelling of the DNA to be analysed.

The kit may contain therefore aliquots with the adapter sequences for one or more restriction enzymes and one or more related universal primers according to the sequence present in the adapter sequences, said primers may be provided in a pre- labelled form with a respective fluorophore. The kit may otherwise comprise labelled nucleotides for use in the PCR reaction in order to label the DNA to investigate and the related PCR.reagents. The kit may also contain one or more restriction enzymes suitable for use as explained above, with the microarray herein described.

In an embodiment said enzymes are chosen between EcoRI and Msel, said adapter sequences are SEQ IDs NOs 1 and 2 for EcoRI and SEQ IDs NOs 3 and 4 for Msel, and said PCR primers are SEQ ID NO 124 and SEQ ID NO 125.

The kit may further comprise computer storing means containing a data base that allows by mans of a computer to identify automatically specific transgenic events or specific GMO. This computer storing mean may be easily made so to provide a virtual

"microarray" (e.g. figure 3) that can be compared to the developed microarray in order to have an automatic reading of the lighted spots on the developed microarray.

The kit may further contain one or more DNA aliquots for use as positive control in PCR reaction. This DNA may be any plant genomic DNA identifiable on the microarray by a related control probe.

The presence of such a control signal will confirm the success of the genomic amplification and the functioning of the labelling. The kit herein described may also comprise a support for the storage and the reading of a software that allows the installation of said software on a computer, in order to carry out the reading of slide by a computer.

Support herein means, for example CD, DVD, tapes, USBP pen, EPROM, floppy disks, hard disks, etc. and/or the software may be downloaded from a network. Alternatively, the kit, may comprise a code, or a password and a login, in order to use the virtual slide for the data reading by connection to a suitable server.

The virtual slide according to the present description is a software allowing the reading of the microarray by a computer and the elaboration of some data related to the detected events.

Said software may carry out the step f. of the indicated method below and will be explained in detail later.

The invention also discloses a method for the simultaneous detection of up to 135 transgenic events comprising the following steps: a. extracting the DNA from a sample comprising the plant material to be analysed, b. digesting said DNA with one or more restriction enzymes, wherein said restriction enzymes do not have restriction sites in the probes of SEQ IDs NOs 5-14, 33, 35-106 and wherein said restriction site is also not comprised in further probes, if present, with control sequences, c. carrying out a ligation reaction between the fragments obtained by said digestion and adaptor sequences comprising a 3' region complementary to the sticky ends generated by said restriction enzymes on said fragments, and a 5' region complementary to a pair of universal primer for PCR amplification, d. carrying out a PCR reaction on the fragments thus obtained using said universal primers optionally labelled with fluorophors or carrying out a PCR reaction on the fragments thus obtained using said universal primers and at least a labelled dNTP, e. hybridising the thus amplified DNA on the microarray according to claim 1 and f. reading the results thus obtained on said microarray.

The genetic material to be analysed can be directly extracted from any part of the plant to be examined if the analysis is to be carried out directly done on a plant, or from plant material to analyse or from alimentary complex matrixes comprising this plant material.

The restriction enzyme or restriction enzymes used for the digestion may be any enzyme having no restriction sites in any of the sequences from SEQ IDs NOs 5 to SEQ IDs NOs 107 (or, when SEQ IDs NOs 15-32 and/or 34, 107 are not used as control sequences, wherein said restriction site is also absent in the selected control sequences). This control is easily carried out due to the disclosure of the sequences in object in the present presentation. The restriction sites analysis may be easily carried out by using suitable softwares such as, by way of example, the webcutter software available on web. EcoRI and/or Msel are an example of these enzymes. Enzymes whose cut generates the so called "sticky ends" are preferred for practical reasons. Said sticky ends have a single strand portion due to enzymatic cut that allows the design of adaptor sequences based on said sticky ends and on the universal primers to be used.

The realisation of the adaptor sequences can be done without difficulties by a skilled person that will design said sequences on the basis of the complementarity to the single strand generated by the cut of the enzyme (protruding end or sticky end) and of the sequence of the universal primer pair to be used. The skilled person will realize that the adaptors will be specifically designed for the single stranded 5' end as well as for the single stranded 3' end. The adaptor sequence may be carried out based on the sequence universal primer to which it has to bind or, vice versa, the universal primer may be carried out based on the adaptor sequence as designed.

It is obvious that the skilled person will carry out universal primer sequences that will not form direct bonds with the sequences blocked on the microarray at the hybridization conditions used.

This is also easily verifiable using software that may be also available on web. The amplification can be carried out with suitable universal primers, pre-labelled with markers reported above that are usable in the microarray technique, or the amplicons may be labelled for example, using at least one labelled dNTP with one of the aforesaid markers that will be incorporated in the amplified sequences during the PCR reaction.

Otherwise, a method of indirect labelling may be used that foresees, for example, the hybridization on the slide of primers as the ones used for the amplification, labelled with suitable markers to the microarray technique.

The present description also discloses a software that allows to carry out by a computer program step f. of the method described.

This software can be loaded on a computer support and may be provided with the kit, or may be accessible by web connection. The software comprises a database concerning the sequences and the events herein described.

In an embodiment, upon opening of the database, it is possible to enter the option "Coordinates of sequences preview" (registry ("Anagrafici") window), that allows to obtain information about the correspondence between spot and sequence and, consequently, on the correspondence between spot and transgenic event as described in Fig.4. The window that appears allows carrying out researches in two directions: a. When a particular transgenic event is expected, it is possible carry out an aimed research by introducing the name of the expected GMO event as classified in the present description (e.g. 5345) and checking in the "COORDINATES" window the sequences (spots) that shall turn on the microarray slide (with the correspondent coordinate), as visualised in figure 5. b. When a particular transgenic event is not expected, but simple identification of the specific GMO is desired on the basis of the spots will turn on, the "RESEARCH" mode has to be entered. A video with three windows appears (on the right hand side of the page in figure 6), corresponding to the three co-ordinates needed for the identification of the sequences corresponding to the spots on the slide: Block, Row, Column (as showed in figure 6):

Upon introduction of the co-ordinates corresponding to the spots that are lighted on the microarray slide it is possible to go back to the transgenic events comprising said sequences and consequently it is possible to characterise the corresponding GMO. Obviously, the same sequence (spot) may belong to several events, but it is from the sum of the various spots that it is possible to achieve the correct characterisation of the event as showed in figure 7.

An aid in this phase of the characterisation of the correct target event is in the section "Preview of the spots corresponding to an event" of the database. In this section is possible to identify the spots corresponding to each transgenic event as showed in figure 8.

By positioning the cursor on each spot it is possible to know to which sequence it corresponds, and upon clicking on the spot the window "Gene's registry" appears, disclosing all the information on the sequence itself as showed in figure 9. The examples reported herein below are provided for a better understanding of the description and they are not intended as a limitation of the claims. EXAMPLES Example 1

Method of genomic DNA amplification A genomic DNA digestion was initially carried out (about lμg), using two different restriction enzymes:

DIGESTION: lμg of genomic DNA, 2.5U EcoRI, 2.5U Msel, Ix Buffer RL (Universal Buffer: TRIS - 10OmM Acetic Acid (Hac) pH 7.5, 10OmM Magnesium Acetate (MgAc), 50OmM Potassium Acetate (Kac), 5OmM Dithiothreitol (DTT), H₂O up to 100ml), H₂O up to 15μl. Incubation at 37°C for 2 hours.

To the pool of the fragments obtained, after the enzymatic digestion, adaptors have been linked by using the T4 Ligase enzyme:

Ligation: to the digestion mixture (15μl of reaction), 5μl of mixture comprising: 0.5 pMol Msel Adaptor (SEQ ID NO:3, SEQ ID NO:4), 1.2 mM ATP, Ix Buffer RL (Universal Buffer), 0.5U DNA Ligase, H₂O up to 20μl have been added. Incubation was carried out at 37°C for 5 hours. After the EcoRI/Msel Adapters ligation a population of fragments has been obtained, with specific adaptors to the 3' and 5' ends. At this point a reaction of amplification is carried out using universal primers designed on the adaptor sequences:

Amplification: 5μl Ligation Mixture, Ix Buffer (INVITROGEN - Taq DNA Polymerase, Recombinant - 10342-020), 1.5mM MgCl₂ (TNVITROGEN - Taq DNA Polymerase, Recombinant - 10342-020), 0.2mM dNTPs, 0.3μM EcoRI Universal Primers (SEQ ID NO:124), 0.3μM Msel Universal Primers (SEQ ID NO:125), IU Taq DNA Polymerase (INVITROGEN - Taq DNA Polymerase, Recombinant - 10342-020), H₂O up to 50μl. Polymerase chain Reaction (PCR) Program: 1 cycle (4minutes 94°C), 35 cycles (1 minute 92°C, 30 seconds 60°C, 1 minute 72°C). In this way all the fragments obtained by the reaction of enzymatic restriction of the genomic DNA, are uniformly amplified. The amplified is used; subject its labelling with fluorophores, for the hybridisation of the microarray slide. Example 2 Amplified DNA labelling and Microarray hybridisation The amplified genomic DNA is labelled with the "BioPrime® Total Genomic

Labeling System" (INVITROGEN - 18097-012) kit following the manufacturer's instructions. The labelled sample is precipitated by Spin - Vacuum Savant and is subsequently re suspended in the hybridisation solution. Microarray hybridisation: The probes have been blocked on the slide for the detection of transgenic events indicated in table 2, in 16 copies per probe. Before carry out the hybridisation reaction, the activation of the slide shall be carried out (glass surface chemistry: EPOXY Surface Coating Slides; spotting buffer: Scott-Nexterion spotting buffer; probe's concentration: 30μM), by the use of a blocking solution (10x Sodium Saline Citrate (SSC), 0.1% Sodium Dodecyl Sulfate (SDS), 0.066 Sodium Tetrahydridoborate (NaBH₄), H₂O up to 50ml). The slide is treated with the blocking solution for 20 minutes to 42°C. Washings are carried out: twice (Sodium Saline Citrate Ix for 5 minutes at room temperature), twice (Sodium Saline Citrate O. Ix for 5 minutes at room temperature).

Prehybridisation:

A solution consisting of: 5x Sodium Saline Citrate (SSC), 0.1% Sodium Dodecyl Sulfate, 200μg Salmon Sperm DNA, 5x Denhardt's Solution (5g Ficoll, 5g

Polyvinyl pyrrolidone, 5g albumin of bovine serum, fraction V, H₂O up to 500ml),

H₂O up to 2ml is prepared. The solution must be filtered by 0.2μm filters. The slide area comprising the probes is delimited with a "frame" (Gene Frame 21x22mm and cover slips - AB 1043 CELBIO). 1 lOμl of prehybridisation solution are placed within this area and the slide is covered with the cover slip. The slide with the prehybridisation solution is incubated at 42°C for 2 hours.

Hybridisation:

A solution consisting of: 5X Sodium Saline Citrate (SSC), 0.1% Sodium

Dodecyl Sulfate, 25% Formamide, 200μg Salmon Sperm DNA, H₂O up to 2ml is prepared. The solution must be filtered by 0.2μm filters and preheated at 42°C. The sample of the amplified and labelled DNA is re suspended in about HOμl of hybridisation solution. The DNA sample, re suspended in hybridisation solution, is denatured at 95°C. The hybridisation solution is placed in the centre of the "frame" that defines the area comprising the target probes, and the cover slip is placed on this area. The slide is hence incubated to 42°C for 16 hours (alternatively it was carried out a different hybridisation at 50°C for 3 hours without Formamide in the hybridization solution).

Post-hybridisation washing:

1^st WASHING - in a volume of 50ml solution: Ix Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulphate (SDS) for 5 minutes to 42°C;

2^nd WASHING - in a volume of 50 ml solution: 0.2x Sodium Saline Citrate (SSC), 0.1% Sodium Dodecyl Sulphate (SDS) for 5 minutes to 42°C;

3^rd WASHING - in a volume of 50ml solution: 0.2x Sodium Saline Citrate (SSC) for 5 minutes to 42°C; 4^th WASHING - in a volume of 50ml solution: 0.2x Sodium Saline Citrate

(SSC) for 5 minutes to 42°C.

The slide has been dried by centrifugation to 800 rpm for 5 minutes.

Example 3

Comparative tests between two methods of amplification pre-hybridization: Comparative tests were carried out between the amplification method of the present description and the "Whole Genome Amplification" method. The comparison has been carried out based on the Southern Blotting technique. This test consisted in the loading of an agarose gel of two samples in six different dilutions (1:10; 1:20; 1:50; 1:100; 1:500.

The first sample was obtained by genomic DNA digestion (Standard FLUKA - monδlO 5%), with two restriction enzymes (EcoRI, Msel) and by ligation of the adaptors to the restriction fragments pool: EcoRI Adapter (SEQ ID NO:1, SEQ ID NO:2), Msel Adapter (SEQ ID NO:3, SEQ ID NO:4). An amplification reaction was carried out using universal primers (SEQ ID NO: 124, SEQ ID NO: 125) designed on the adapters sequence (SEQ ID NO: I₅ SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4). All the protocol is described in the previous examples. The second sample was produced by carrying out genomic DNA amplification with a Whole Genome Amplification kit (GenomiPhi VE Amplification Kit - 25-6600-30 - GE Healthcare) following the manufacturer's instructions. Further, a positive control (PCR product obtained by amplification of the sample DNA using the same primers needed for the hybridization probe production) and a negative control (Standard FLUKA - mon810 0%) were loaded. The 0.7% agarose gel was placed in an electrophoresis cassette and was ran "over night" with a low voltage (~ 20V).

The comparison was carried out using a probe consisting of an amplicon obtained by genomic DNA amplification of MON810 5% (Standard FLUKA - mon810 5%), with specific primers for the amplification of the border region between the host plant genome and the target gene Cryl Ac that confers resistance to European corn borer (Ostrinia nubilalis) to the corn mon810 [Hernandez M. et al. (2003) A specific real — time quantitative PCR detection system for event MON810 in maize YealdGard based on the 3' - transgene integration sequence. Transgenic Research 12: 179-189]. The selected probe is highly specific for the transgenic event MON810.

Result: The Southern blot has showed the presence of the target band in the DNA sample amplified by Whole Sample Amplification (digested with the EcoRI/Msel restriction enzymes), but the band is not present in the DNA sample amplified by the Whole Genome Amplification kit (GenomiPhi VE Amplification Kit - 25-6600-30 - GE Healthcare) (Figure 1 and 2).

Therefore the testing on slide was carried out for testing the same single event on microarray using 9 probes of the invention (each of these probes is repeated 16 times) to detect the transgenic event: mon810. The tests carried out showed equivalence between the digestion - amplification method as described above and a standard amplification using nine different couples of primers specific for each probe blocked on the slide of the experiment - Table 3).

Example 4

Microarray probe design:

A database comprising sequences and information on target genes, promoters, terminators used for the realisation of 157 transgenic events has been designed. Based on this collected information, 102 sequences were selected for the design of probes to block on the definitive slide (9 of this sequences were used for the design of 9 probes that were blocked on the microarray slide for the detection of the single transgenic event MON810 that was used for the comparison between the methods of the example 3), for the detection of 136 different transgenic events of agro- alimentary importance.

Probes characteristics: a. designed taking into account of the restriction sites for the enzymes used for the genomic DNA digestion (EcoRI, Msel), so to not comprise such sites; b. they are 50mer (oligonucleotide of a 50bp length); c. they have an "annealing temperature" of about 80°C

The probes obtained are hence used in similarity searches (public database used), in order to verify the specificity thereof. This work has allowed to obtain high specific probes for the detection of the single transgenic events and suitable for a detection using the amplification method herein developed (Table in the Section IX).

The sequences of the selected probes are indicated as:

SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10,SEQ ID NO:11 ,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:21 ,SEQ ID NO:22,SEQ ID NO:23,SEQ ID NO:24,SEQ ID

NO:25,SEQ ID NO:26,SEQ ID NO:27,SEQ ID NO:28,SEQ ID NO:29,SEQ ID NO:30,SEQ ID NO:31 ,SEQ ID NO:32,SEQ ID NO:33,SEQ ID NO:34,SEQ ID NO:35,SEQ ID NO:36,SEQ ID NO:37,SEQ ID NO:38,SEQ ID NO:39,SEQ ID NO:40,SEQ ID NO:41 ,SEQ ID NO:42,SEQ ID NO:43,SEQ ID NO:44,SEQ ID NO:45,SEQ ID NO:46,SEQ ID NO:47,SEQ ID NO:48,SEQ ID NO:49,SEQ ID NO:50,SEQ ID NO:51 ,SEQ ID NO:52,SEQ ID NO:53,SEQ ID NO:54,SEQ ID NO:55,SEQ ID NO:56,SEQ ID NO:57,SEQ ID NO:58,SEQ ID NO:59,SEQ ID NO:60,SEQ ID NO:61 ,SEQ ID NO:62,SEQ ID NO:63,SEQ ID NO:64,SEQ ID NO:65,SEQ ID NO:66,SEQ ID NO:67,SEQ ID NO:68,SEQ ID NO:69,SEQ ID NO:70,SEQ ID NO:71 ,SEQ ID NO:72,SEQ ID NO:73,SEQ ID NO:74,SEQ ID

NO:75,SEQ ID NO:76,SEQ ID NO:77,SEQ ID NO:78,SEQ ID NO:79,SEQ ID NO:80,SEQ ID NO:81 ,SEQ ID NO:82,SEQ ID NO:83,SEQ ID NO:84,SEQ ID NO:85,SEQ ID NO:86,SEQ ID NO:87,SEQ ID NO:88,SEQ ID NO:89,SEQ ID NO:90,SEQ ID NO:91 ,SEQ ID NO:92,SEQ ID NO:93,SEQ ID NO:94,SEQ ID NO:95,SEQ ID NO:96,SEQ ID NO:97,SEQ ID NO:98,SEQ ID NO:99,SEQ ID NO:100,SEQ ID NO:101 ,SEQ ID NO:102,SEQ ID NO:103,SEQ ID NO:104,SEQ ID NO:105,SEQ ID NO:106,SEQ ID NO:107

Each of the elements specifically indicated above and below, hence any probe may be individually excluded and/or replaced from an equivalent in the carrying out of the present invention without changing the scope thereof.

DESCRIPTION OF THE SEQUENCES

TABLES

Table 2, below, shows the list of all the events detectable with microarray herein described and the organisms in which these events are detectable at present.

Table 3, that follows, shows the relation between 15 events indicated as particularly interesting in the description, the gene coding thereof and the pool of sequences that identify each of the 15 events.

Table 4 that follows shows the relationship between the 135 events shown in the description, the genes coding for them and the set of sequences that identify each of the 135 events.

It is understood that a COMBINATION is herein a set of sequences that, as a whole, allows the detection of a specific transgenic event. Despite the fact that a same sequence may appear in different COMBINATIONS each COMBINATION is unique hence consists of a set of sequences forming a single set. Each COMBINATION (single set of sequences) is indicated with a progressive number from 1 to 95 to indicate that the totality of the sequences (probes), present on the microarray slide, allows obtaining 95 different unique COMBINATIONS that at the same time will allow the detection of 135 different transgenic events. It follows that combination 95, constituted of the sequences SEQ ID NO: 104, 80, 43, 30, 56, is unique in its composition of sequences and in the ability of identify the MXB- 13 transgenic event.

BIBLIOGRAFY

Min W, Wen-Li M, Bao Z, Ling L, Zhao-Hui S, Wen-Ling Z. Oligonucleotide microarray with RD-PCR labeling technique for detection and typing of human papillomavirus. Curr Microbiol. 2006 Mar;52(3):204-9; Mo XY, Ma WL, Li L, Xu QL, Zhang YL, Zheng WL. The effects of different sample labelling methods on signal intensities of a 60-mer diagnostic microarray. J Virol Methods. 2006 Jun;134ri-2):36;

Li L, Ma WL₃ Zhu J, Shi R, Liu CH, Chen JK, Zheng WL. A modified restriction display PCR method in sample-labelling of DNA microarray. J Virol Methods. 2003 Dec;l 14(11:71-5;

Christer Røss Nielsen, Knut G. Berdal and Arne Hoist- Jensen. Anchored PCR for possible detection and characterisation of foreign integrated DNA at near single molecule level. European Food Research and Technology Published online: 25 April 2007; Detection and Traceability Technologies to Underpin the GM Inspectorate T.

R. Allnutt, http://www.gminspectorate.gov.ulc/documents/Detection_and_traceability_report_01 1205.pdf;

Arkiditis et al, Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection method Electronic Journal of Biotechnology ISSN: 0717-3458. vol 11, No 2, 15 April 2008;

US 6,270,966

US 20070117106Al

Claims

1. Microaxray comprising, as probes attached on the surface of said microarray, nucleotide sequences comprised in the group of from SEQ ID NO 5 to SEQ ID NO 107, said microarray allowing the simultaneous identification of the presence or absence of each of at least 15 transgenic events in samples comprising plant material.

2. Kit for the identification of the presence or absence of transgenic events in samples comprising plant material, comprising one or more arrays according to claim 1.

3. Kit, according to claim 2, further comprising one or more aliquots of one or more restriction enzymes and/or one or more aliquots of an adaptor sequence for each of said restriction enzymes and/or one or more aliquots of one or more PCR primer capable of annealing to each of said adaptor sequences in a PCR reaction.

4. Kit, according to claim 3 wherein said enzymes are selected between EcoRI and Msel, said adaptor sequences are SEQ IDs NOs 1 and 2 for EcoRI and

SEQ IDs NOs 3 and 4 for Msel, and said PCR primers are SEQ ID 124 and SEQ ID 125.

5. Kit for OGM identification in samples comprising plant material, comprising one or more microarray according to claim 1 and one or more aliquots for the detection of said probes.

6. Kit, according to claim 5, further comprising one or more aliquots of one or more restriction enzymes and/or one or more aliquots of an adaptor sequence for each of said restriction enzymes and/or one or more aliquots of one or more PCR primer capable of annealing to each of said adaptor sequences in a PCR reaction.

7. Kit, according to claim 6 wherein said enzymes are selected between

EcoRI and Msel, said adaptor sequences are SEQ IDs NOs 1 and 2 for EcoRI and SEQ IDs NOs 3 and 4 for Msel, and said PCR primers are SEQ ID 124 and SEQ ID 125.

8. Kit, according to any one of claims 2 to 7 further comprising a computer readable support for the storage of a computer software, said support comprising a software for the reading of said microarray by computer means.

9. Kit, according to any one of claims 2 to 7 further comprising an identifying code for the accession to a software for the reading of said microarray by computer means via a network access.

10. Method for the simultaneous detection of the presence/absence, and the identification, when present, of at least 15 transgenic events comprising the following steps: a. extracting the DNA from a sample comprising the plant material to be analysed, b. digesting said DNA with one or more restriction enzymes, wherein said restriction enzymes do not have restriction sites in the probes of SEQ IDs NOs 5-14, 33, 35-106 and wherein said restriction site is also not comprised in further probes, if present, with control sequences, c. carrying out a ligation reaction between the fragments obtained by said digestion and adaptor sequences comprising a 3 ' region complementary to the sticky ends generated by said restriction enzymes on said fragments, and a 5' region complementary to a pair of universal primer for PCR amplification, d. carrying out a PCR reaction on the fragments thus obtained using said universal primers optionally labelled with fluorophores or carrying out a PCR reaction on the fragments thus obtained using said universal primers and at least a labelled dNTP, e. hybridising the thus amplified DNA on the microarray according to claim 1 and f. reading the results thus obtained on said microarray.