RU2453605C2 - Differentiating and specific oligonucleotides for identification of dna sequences of transgene plants in foodstuff, method for identifying transgene products, biochip, combination of oligonucleotides (versions) and kit for implementing such method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for identifying DNA sequences in a biological material involves recovery of nucleic acids, carrying out a multi-primer symmetric polymerase chain reaction with using DNA recovered from the analysed material that is followed by hybridisation of fluorescent marked or biotin-modified reaction products on the specialised biochips. There are presented a oligonucleotide kit, a biological microchip containing up to 11 types of differentiating oligonucleotides and a reagent kit for implementing such method, and also versions of the formulations of combined nucleotides for implementing such method.

EFFECT: invention allows more rapid foodstuff analysis for genetically modified ingredients and higher accuracy.

18 cl, 5 dwg, 6 tbl, 8 ex

Description

Technical field

The invention relates to the field of biotechnology, molecular biology and genetic engineering, and in particular to a method for identifying DNA sequences of genetically modified sources and food products based on them, including the steps of DNA amplification and subsequent hybridization of the amplified DNA on a biological microchip. The invention also discloses the composition of highly specific DNA probes and oligonucleotides for PCR, a set of reagents for identification.

State of the art

The development of diagnostics of genetically modified sources (GMI) in food is currently an urgent task. Directive No. 1829/2003 of the European Parliament and of the Council of September 22, 2003 on genetically modified food and feed [1] introduced new labeling rules in the EEC countries from April 2004 on which a GMI threshold level of 0.9% is established. The introduction of a 0.9% threshold level is associated with the sensitivity of the method of determining GMI and the accidental ingestion of GMI into food products. Mandatory labeling of food products from GMI is introduced in Russia.

Known methods for determining GMI in food products. One of the methods is based on the use of enzyme-linked immunosorbent assay using appropriate antibodies to proteins that make up genetically modified plants [2].

A more stable and widespread method of identification is based on the analysis of DNA contained in transgenic plants using polymerase chain reaction (PCR) [3, 4, 5, 6, 7].

At present, GOST 52173-2003 is in force in the Russian Federation for the determination of GMOs in food products and plant materials by PCR [8]. In the process of PCR, DNA extracted from plant materials and pairs of synthetic primers are used. After the amplification reaction, the reaction products are separated by gel electrophoresis and detected by staining them in a gel with a fluorescent dye. Amplified DNA fragments containing a fluorescent label are identified either after the amplification reaction or in real time by measuring the intensity of the fluorescent signal.

Recently, biochips have been used more and more widely, which make it possible to simplify the GMI identification procedure [9, 10, 11]. Currently, GOST 52174-2003 is in force for the determination of GMI in food products and plant materials using microchips [12]. The issues of improving the accuracy and reliability of the determination of GMI using biochips are usually solved by improving any one procedure in the multi-stage process of creating a biochip or in the process of hybridization and scanning of the results. Nevertheless, the known technical solutions have a number of disadvantages that are associated either with an unsatisfactory choice of gene regions, or with the choice of parameters of multi-primer PCR, or with the design of a biochip, for example, with the arrangement of clusters on the working field of the biochip.

One of the disadvantages of the known methods for identifying GMIs is a fairly narrow set of genetic markers and corresponding primers for determining the presence of transgenic plant markers in the process of identifying GMIs. In one variant of the quantitative identification of GMI, RRS, Bt-11, 35S CMV markers are used, and the pectin gene is used as a reference marker [3]. In another embodiment [14], HMG, RRS02 markers are used; in the application [13], EPSPS, cry1A (b), cry9C and PAT markers are used.

The invention is known [15], in which from 3 to 10 pairs of primers are used to determine the presence of GMI for carrying out a multi-primer PCR procedure. Genetic markers are selected from the group of P35S, TNOS, PNOS, NPTII, GUS, wild type EPSPS gene, modified type EPSPS gene, CP4, vignatrypsase CpTI, and antibiotic peptide shiva gene. Amplification products hybridize on a membrane equipped with specific probes, and hybridization results are detected. The disadvantage of the invention is the fact that in the list of markers there are no markers of positive and negative control.

The choice of parameters of multi-primer PCR largely determines the effectiveness and stability of the diagnosis. Known works in which to increase the accuracy of the procedure include additional enzymatic treatment. The invention is known [17], which describes a method for amplification of DNA using a reagent composition that contains thermostable enzyme exhibiting DNA polymerase activity as the main component, and the thermostable Archeoglobus fulgidus enzyme exhibiting 3'-exonuclease activity as the minor component. but devoid of DNA polymerase activity. According to the invention, the enzyme is able to interact with Taq polymerase as a correcting enzyme in order to remove erroneously paired ends of primers that lead to premature shutdowns, as well as to free ends of primers that are more efficiently elongated by polymerase, to correct base incorporation errors and enable polymerase form extended PCR products. Using this composition improves the accuracy of the amplification process compared to using only the main enzyme.

The known method [18], which sets forth the principle of processing primers obtained by PCR using lambda exonuclease or gene product of T7 phage 6 for 15 min at a temperature of 37 ° C. As a stopping agent, a 10 mM EDTA solution is used. This invention does not address the issues of processing exonuclease products obtained in the process of multi-primer PCR, and the fact of processing exonuclease

A known method [19], which considers the simultaneous multiple amplification of nucleic acids and the use of exonuclease. According to the invention, the method comprises: (a) contacting a nucleic acid with one or more pairs of primers under conditions that allow annealing of the primers with nucleic acid, wherein each primer has a double-sided structure AB, in which part A is specific for the target nucleic acid sample sequences acid, and part B is a constant sequence common to all primers; (b) conducting a first amplification reaction; (c) treating the double-sided primers or products of the first amplification reaction with an exonuclease; (d) contacting the products of the first reaction with primers containing part B under hybridization conditions and carrying out a second amplification reaction. In the present invention, the Bt176, Bt11, Mon810, Amp, Nos, 35S, IPC, Corn ref genes are used as target sequences. Exonuclease I was selected as the enzyme. The disadvantage of this method is the need for two-stage PCR.

Known kits [20], which are produced by CJSC BioChemMak, which include: a reaction mixture for the detection of GMI (2 × 50), a reference mixture, DNA calibrators of the plant gene and GMI, positive control. The kit includes one of the genes that make up the Roundup Redi group, Bt176 Maximizer, Bt11, LibertyLink, YelDKard MON810, StarLink, GA21, ITC 1507, LL (T25). Using the kits, a GMI is quantitatively analyzed by real-time PCR (GMO-Quant kits). The invention is known [15], which describes a kit for the detection of GMOs, which includes primers and probes for conducting multiplex PCR and hybridization. The closest analogue of the claimed invention in terms of the kit, which includes primers and reagents for creating reaction mixtures, is described in the guidelines of MUK 4.2.2008-05 [16].

The closest analogue in terms of the method is the invention [9], which uses genetic markers selected from the group: 35S — the promoter region of the cauliflower mosaic virus, the gus marker gene from the bacterium Escherichia coli, the terminator region of the nos gene from Agrobacterium tumefaciens to identify GMIs , the nptII marker gene from the Tn5 transposon, the terminator portion of the ocs gene from Agrobacterium tumefaciens, which are included in the guidelines [16].

A method for identifying transgenic DNA sequences in plant material and products based on such material [9] includes DNA analysis using polymerase chain reaction (PCR) and recording the results of the analysis, and PCR is carried out on the DNA preparation as a multiplex reaction to obtain a fluorescently labeled product, this uses specific pairs of primers, one of which is fluorescently labeled and is present in excess relative to the second unlabeled primer, the PCR results are analyzed using a hybrid biochip and register using a hardware-software complex that allows you to display the fluorescence of biochip cells on a computer screen. However, the invention contains a limited number of primers, and the enzyme is not included in the kit to increase the efficiency of the analysis when using multi-primer symmetric PCR.

The technical task of the invention is to create a more advanced diagnosis of GMI on the basis of a new method using a new biochip with better characteristics at a low cost of screening GMI, and on the basis of a set for diagnostics, which includes new primers, a new biochip and enzyme for processing PCR products.

Another task is to increase the reliability and reproducibility of the results. This problem is solved by using new, more efficient primer sequences in biological GMI diagnostics chips and by developing a method for processing products of multi-primer symmetric PCR with an enzyme that increases hybridization efficiency, as well as by arranging positive control clusters that simultaneously act as identifiers of the boundaries of the biochip working area .

SUMMARY OF THE INVENTION

The proposed diagnostic method is based on carrying out symmetric multi-primer PCR using specific primers complementary to the sequences of GMI marker genes, and subsequent hybridization of the PCR products on a biochip containing an original set of differentiating oligonucleotides.

One of the objects of the invention is a method for identifying markers of genetically modified plants, including providing a sample with the studied DNA, direct and reverse primers specific for DNA belonging to the genes of GMI markers, and a biochip, conducting multi-primer PCR in the presence of a sample with the studied DNA and specific primers for to the indicated GMI markers, treatment of PCR reaction products with an enzyme, incubation of the PCR product with differentiating oligonucleotides immobilized on the surface of the biochip under conditions x hybridization, determination of the presence of GMI by detection of the formed hybridization complexes on a biochip. In this method, the biochip contains more than 6 types of differentiating oligonucleotides selected from the sequences shown in SEQ ID NO: 23-33, taking into account the high specificity requirements for the conserved regions of GMI genes included in the list shown in table 1. As specific primers, use primers, the sequence of which is selected from the group consisting of SEQ ID NO: 1-22, as a PCR, use a multi-primer symmetric PCR, the products of which are preferably treated in a volume of 20 μl, preferably 1 μl a solution of phage lambda exonuclease in a concentration of from 5 to 20 units of activity and incubated for from 30 minutes to 90 minutes, preferably at a temperature of 37 ° C. In a pair of forward and reverse primers, one primer contains a label that, after hybridization, DNA is detected, registered and used to identify GMI markers, and the differentiating oligonucleotide SEQ ID NO: 36, partially complementary to the oligonucleotide SEQ ID NO: 8, is used as a negative control. as a positive control, the differentiating oligonucleotide SEQ ID NO: 34 is used, to which the specific oligonucleotide SEQ ID NO: 35 is complementary.

Another object of the invention is a specific oligonucleotide for use in a method for identifying genetically modified plant markers according to claims 1 to 4, which is selected as a primer of a symmetric multi-primer PCR and which is characterized by a nucleotide sequence selected from the sequences shown in SEQ ID NO: 4, 7, 8, 11-14, 21, 22.

The next object of the invention is a differentiating oligonucleotide as a probe for identifying genetically modified plant markers for use in the GMI identification method according to claims 1 to 4, which is characterized by a nucleotide sequence selected from the sequences shown in SEQ ID NO: 24, 28, 29 , 33.

Another object of the invention is a combination of oligonucleotides to identify a NOS marker for identifying a 3'-untranslated region of the nopalin synthase gene, including oligonucleotides with the sequences SEQ ID NO: 24 and SEQ ID NO: 3 and SEQ ID NO: 4.

Another object of the invention is a combination of oligonucleotides to detect an NPTII marker encoding neomycin phosphotransferase II, comprising oligonucleotides with the sequences SEQ ID NO: 26 and SEQ ID NO: 7, and SEQ ID NO: 8.

Another object of the invention is a combination of oligonucleotides for detecting the Lectin marker represented by the Le1 gene family of Glycine max, comprising oligonucleotides with the sequences SEQ ID NO: 28 as a probe and two specific SEQ ID NO: 11 and SEQ ID NO: 12.

Another object of the invention is a combination of oligonucleotides for detecting the Zein marker represented by the zein gene family of Zea Mays, comprising oligonucleotides with the sequences SEQ ID NO: 29 and SEQ ID NO: 13 and SEQ ID NO: 14.

Another object of the invention is a combination of oligonucleotides for detecting the Patatin marker represented by the pat1 gene family of Solanum tuberosum encoding a patatin precursor comprising oligonucleotides with the sequences SEQ ID NO: 33 and SEQ ID NO: 21 and SEQ ID NO: 22.

Another object of the invention is a biochip for identifying GMI markers, which is a supporting element on which differentiating oligonucleotides in the form of probes forming clusters are immobilized, characterized in that more than 6 types of clusters with differentiating oligonucleotides selected from SEQ ID NO are immobilized on its surface -33, wherein the biochip contains at least one cluster comprising a positive control oligonucleotide shown in SEQ ID NO: 34, and at least one cluster comprising o negative control ligonucleotide shown in SEQ ID NO: 36.

Another object of the invention is a kit for identifying GMI markers, including:

a) at least one biochip for a one-time diagnosis, containing more than 6 types of differentiating oligonucleotides specific for identifiable markers of transgenic plants, with the sequences shown in SEQ ID NO: 23-33, one or more differentiating oligonucleotides as a positive control, with the sequence shown in SEQ ID NO: 34 and one or more differentiating oligonucleotides as a negative control, with the sequence shown in SEQ ID NO: 36;

b) specific oligonucleotides used as PCR primers to identify GMI markers whose sequences are presented in SEQ ID NO: 1-22, and a specific oligonucleotide shown in SEQ ID NO: 35, which is complementary to the differentiating oligonucleotide of the positive control SEQ ID NO: 34 placed on a biochip in clusters of positive control;

c) reagents that are selected from the group consisting of: reagents for sample preparation, reagents for conducting multi-primer symmetric PCR, reagents for hybridization, or combinations thereof and an exonuclease of phage lambda.

List of drawings

Figure 1 - image of clusters in the identification of a sample of a food product containing GMI.

Figure 2 is a variant of the arrangement of clusters on the biochip, where 1 is a negative control cluster, 3, 13, 15 are positive control clusters, 2 is a Patatin cluster, 4 is an Npt II cluster, 5 is a 35S cluster, 6 is a Gus cluster, 7 - Lectin cluster, 8 - Nos cluster, 9 - Zein cluster, 10 - Cry1A cluster, 11 - Bar cluster, 12 - Ocs cluster, 14 - EPSPS.

Figure 3 - hybridization results when checking the specificity of differentiating primers, where a) -l) checking hybridization to differentiating primers 35S, Nos, Ocs, NptII, Gus, Lectin, Zein, Patatin, Bar, Cry1A, EPSPS, respectively.

Figure 4 - comparison of the efficiency of hybridization for different methods of processing PCR products, where a) the sample was processed with an exonuclease before hybridization, b) the sample was subjected to thermal denaturation, c) the sample was not processed.

5 is an image of clusters displaying the results of a multi-primer symmetric PCR and hybridization of the biochip when identifying markers: bar, cry1Ab, EPSPS, Patatin.

Description of the invention

A method for identifying GMO markers, in the general case, includes the following stages: selection of markers, selection of primers and probes, synthesis of probes and primers, preparation of a biochip (for example, surface modification and application of probes), DNA extraction, DNA amplification using multi-primer symmetric PCR, processing PCR of the product with an exonuclease, DNA hybridization on a biochip, detection, registration of the obtained result and interpretation of the obtained result.

Marker Selection

In the process of studying methods for diagnosing GMI and known diagnostic biochips [9, 10], it was found that most of the known solutions related to the choice of markers for diagnosis do not take into account the fact that recently there has been a rapid increase in the number of products containing genetically modified plants obtained by different technologies, which leads to the possibility of unreliable detection of food products containing GMI.

The non-obvious solution in the framework of the present invention can be attributed to the selection of a list of markers with the help of which a wider range of transgenic plants is identified than in the known analogues patents. The list of markers presented in table 1, is made in such a way that eliminates the disadvantages of the known inventions. To the list, in conjunction with well-known markers, namely the 35S promoter region of cauliflower mosaic virus, the gus gene from the bacterium Escherichia coli, the terminator region of the nos gene from Agrobacterium tumefaciens, the marker gene npt II from the transposon Tn5 Escherichia coli, the terminator region of the octopinta gene Agrobacterium tumefaciens, which are included in the guidelines, includes additional markers, namely the marker bar, representing the gene from Streptomyces hygroscopicus, which encodes a phosphotricin-acetyltransferase; marker cry1Ab, representing the insect Bt-toxin gene from Bacillus thuringiensis; EPSPS marker, representing the 5-enolpyruvil-chymate-3-phosphate synthase gene from Agrobacterium tumefaciens (CP4), which provides Roundup herbicide resistance; a Patatin marker representing the gene (pat1) from Solanum tuberosum (potato), which encodes a patatin precursor; a Zein marker representing a gene of the zein family from Zea Mays (corn); Lectin marker, representing the Le1 family gene from Glycine max (soy).

Such a combination of markers allows to realize the technical task of the invention related to increasing the efficiency of diagnostics, at the same time, quickly and at minimal cost, diagnose from 1 to 8 GMI markers in one analysis, based on the identification of DNA regions specific to GMI sources and three markers that are widely common plant species such as soy, corn and potatoes.

The selection of specific and differentiating oligonucleotides and their possible combinations

Selection of specific oligonucleotides for PCR

The primers were selected taking into account the requirements of high specificity for conserved genes of the studied GMI objects, which allows one to selectively amplify a DNA fragment of a transgenic plant directly from an isolated biological sample. When choosing primers, the requirements standardly applied to primers are taken into account, which include: the absence of highly stable secondary structures, a small difference in melting points not exceeding 2-3 ° C, and the choice of primer lengths in the range from 15 to 55 n.a.

For the preparation of primers, we used sequences characteristic of the identified transgenic plant and presented in the GenBank database of nucleotide sequences (http://ncbi.nlm.nih.gov), in which the most conserved regions were selected that were not previously used and were not obvious for the preparation of specific and differentiating oligonucleotides to create biological chips.

Each pair of oligonucleotides for PCR specific for the GMI marker consists of one unlabeled (not containing any labels necessary for subsequent analysis and interpretation of the results) specific oligonucleotide from the listed sequences with any odd number with SEQ ID NO: 1 through SEQ ID NO: 21 and labeled (containing one or more labels necessary for subsequent analysis and interpretation of the results) a specific oligonucleotide from the evenly listed sequence numbers with SEQ ID NO: 2 p SEQ ID NO: 22, follows immediately after the first couple specific oligonucleotide.

The selection of differentiating oligonucleotides

Differentiating oligonucleotides were selected taking into account the requirements of high specificity for conserved regions of genes of the studied GMI, which allows selective hybridization of DNA fragments amplified with specific oligonucleotides by PCR directly from biological samples, as well as on the basis of the absence of highly stable secondary structures, ranging in length from 15 to 55 n.a., preferably from 20 to 28 n.a.

When choosing differentiating oligonucleotides, one of the selection criteria was the presence of homology only between each specific oligonucleotide and the corresponding marker. Nevertheless, computer-aided methods for analyzing DNA sequences do not guarantee the possibility of hybridization of a differentiating oligonucleotide specific for one marker being analyzed with DNA of other marker being analyzed, for example, due to the possible formation of complex secondary structures by amplicons. Therefore, to check the specificity of differentiating oligonucleotides, a possible non-specific background was determined during DNA hybridization of each of the 35S, Nos, Ocs, Npt II, Gus, Lectin, Zein, bar, cry1Ab, EPSPS and Patatin markers individually.

For this, by means of PCR with primers specific to only one of the markers, DNA fragments of each marker were individually generated. The obtained samples were treated with an exonuclease and hybridization and washing were performed as described in Example 6. The hybridization results of the specificity testing of differentiating primers are shown in FIG. 3, where FIG. 3, A) for the 35S marker, FIG. 3, B) for the Nos marker, Figure 3, B) for the Ocs marker, Figure 3, D) for the Npt II marker, Figure 3, D) for the Gus marker, Figure 3, E) for the Lectin marker, Figure 3, G) for the Zein marker , Fig. 3, H) for the Patatin marker, Fig. 3, I) for the Bar marker, Fig. 3, K) for the Cry1A marker, Fig. 3, L) for the EPSPS marker.

Based on the analysis of the results of hybridization, the maximum value of the nonspecific signal was established. With this in mind, a negative control oligonucleotide (SEQ ID NO: 36) was compiled, partially complementary to the oligonucleotide sequence of SEQ ID NO: 8. The signal level during hybridization of the fluorescent-labeled oligonucleotide SEQ ID NO: 8 contained in the PCR mixture with the oligonucleotide SEQ ID NO: 36 immobilized on a chip in negative control clusters significantly exceeds the maximum possible level of “nonspecific” hybridization signals and serves as a background, exceeding which is considered as positive signal in the analysis.

Combinations of Oligonucleotides

The following combinations of oligonucleotides are sets of two primers, i.e. specific oligonucleotides, and one oligonucleotide used as an immobilized probe on the surface of the biochip, i.e. differentiating oligonucleotide. The following combinations are used to diagnose GMI within the framework of the present invention:

1. For specific identification of the 35S marker, mainly for identification of the 35S promoter from cauliflower mosaic virus, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 23 as a probe and two specific SEQ ID NO: 1 and SEQ ID NO: 2 oligonucleotides used as PCR primers.

2. For specific identification of the NOS marker, mainly for identification of the 3 'untranslated region of the nopaline synthase gene (NOS), which is part of the Agrobacterium tumefaciens Ti plasmid, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 24 as a probe and two specific SEQ IDs NO: 3 and SEQ ID NO: 4 oligonucleotides used as PCR primers.

3. For specific detection of the OCS marker, mainly for identification of the 3 'untranslated region of the octopinsynthase gene (OCS), which is part of the Agrobacterium tumefaciens Ti plasmid, a combination of one differentiating oligonucleotide SEQ ID NO: 25 as a probe and two specific SEQ IDs is used NO: 5 and SEQ ID NO: 6 of the oligonucleotide used as PCR primers.

4. For specific identification of the NPTII marker, mainly for identification of the gene (neo, nptII) from E. coli encoding neomycin phosphotransferase II, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 26 as a probe and two specific SEQ IDs NO: 7 and SEQ ID NO: 8 of the oligonucleotide used as PCR primers.

5. For specific identification of the GUS marker, mainly for identification of the gene (uidA, GUS) from E. coli encoding beta-glucuronidase, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 27 as a probe and two specific SEQ ID NO : 9 and SEQ ID NO: 10 of the oligonucleotide used as PCR primers.

6. To specifically identify the Lectin marker, mainly to identify the Le1 family gene from Glycine max (soy) that encodes a lectin, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 28 as a probe and two specific SEQ ID NO: 11 and SEQ ID NO: 12 oligonucleotides used as PCR primers.

7. For specific identification of the Zein marker, mainly for identification of the zein family gene from Zea Mays (corn), which encodes zein, use a combination that includes one differentiating oligonucleotide SEQ ID NO: 29 as a probe and two specific SEQ ID NO: 13 and SEQ ID NO: 14 of the oligonucleotide used as PCR primers.

8. For the specific identification of the bar marker, mainly for identification of a gene from Streptomyces hygroscopicus encoding a phosphotricin acetyltransferase, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 30 as a probe and two specific SEQ ID NO: 15 and SEQ ID NO : 16 oligonucleotides used as PCR primers.

9. For the specific identification of the cry1Ab marker, mainly for identification of a gene from Bacillus thuringiensis encoding a Bt toxin, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 31 as a probe and two specific SEQ ID NO: 17 and SEQ ID NO : 18 oligonucleotides used as PCR primers.

10. For the specific identification of the EPSPS marker, mainly for identification of a gene from Agrobacterium tumefaciens (CP4) encoding 5-enolpyruvyl shikimate-3-phosphate synthase, a combination of one differentiating oligonucleotide SEQ ID NO: 32 as a probe and two specific SEQ IDs is used NO: 19 and SEQ ID NO: 20 of the oligonucleotide used as PCR primers.

11. For the specific identification of the Patatin marker, mainly for the identification of the pat1 gene from Solanum tuberosum (potato) encoding the patatin precursor, a combination is used that includes one differentiating oligonucleotide SEQ ID NO: 33 as a probe and two specific SEQ ID NO: 21 and SEQ ID NO: 22 oligonucleotides used as PCR primers.

The following is a list of specific nucleotides (table 2) and a list of differentiating oligonucleotides (table 3) used in the framework of the present invention.

table 2 Specific oligonucleotides for species identification ID SEQ No Gene name Nucleotide sequence one 35S promoter from cauliflower mosaic virus GCGTCATCCCTTACGTCAGTGGAG 2 CATTGCGATAAAGGAAAGGCCATC 3 3 'untranslated region of the nopalin synthase (NOS) gene isolated from Ti plasmid Agrobacterium tumefaciens AACCCATCTCATAAATAACGTCATGCA four AGCAGATCGTTCAAACATTTGGCA 5 3 'untranslated region of the octopin synthase gene (OCS) isolated from Ti plasmid Agrobacterium tumefaciens AGATATGCGAGACGCCTATGATCG 6 GCGGTAAGGATCTGAGCTACACATG 7 gene (neo, nptII) from E. coli encodes neomycin phosphotransferase II CCTGTCATCTCACCTTGCTCCTG 8 GATGTTTCGCTTGGTGGTCG 9 gene (uidA, GUS) from E. coli encodes beta-glucuronidase TTGGAAACGGCAGAGAAGGTACTG 10 CCAGCCATGCACACTGATACTCTTC eleven Glycine max (soy) Lectin gene of the Le1 family, encodes a lectin CGGAATTAACGTCAATTCTATCAGATCC 12 CAGAGAACCCTATCCTCACCCACTC 13 Zein gene of the zein family from Zea Mays (corn), encodes zein ATTATTCCACAATGCTCACTTGCTCC fourteen ATGGCTAGGTGGCTCAGTGCTG fifteen bar gene from Streptomyces hygroscopicus encodes phosphotricin acetyltransferase GGCACGCAACGCCTACGACT 16 GTCCAGCTGCCAGAAACCCAC 17 cry1Ab gene from Bacillus thuringiensi encodes a Bt toxin AGCCAAGACCTCGAGATTTACCTGA eighteen AGGAGAAGTGGTGGCTGTGGTG 19 EPSPS gene from (Agrobacterium lumefaciens CP4), encodes 5-enolpyruvyl shikimate-3-phosphate synthase AATCCTCTGGCCTTTCCGGAAC twenty TATTGATGACGTCCTCGCCTTCC 21 gene (pat1) from Solanum tuberosum (potato), encodes a patatin precursor GTTGCTCTCATTAGGCACTGGCAC 22 TTAATGCATTTTCTTGAACCCTGAGG

Table 3 Differentiating oligonucleotides for species identification of eight GMI marker genes and three soy, potato, and corn markers ID SEQ No Gene name Nucleotide sequence 23 35S mosaic virus promoter CATCTTTGGGACCACTGTCGGCAGAGGCATCTT C cauliflower 24 3 'untranslated region of the nopalin synthase (NOS) gene isolated from Ti plasmid Agrobacterium tumefaciens TGATAATCATCGCAAGACCGGCAACAGGATTCA 25 3 'untranslated region of the octopin synthase gene (OCS) isolated from Ti plasmid Agrobacterium tumefaciens CATGATATTTGCTTTCAATTCTGTTGTGCACGTTGTAAAA 26 gene (neo, nptII) from E. coli encodes neomycin phosphotransferase II GGCTGCATACGCTTGATCCGGCTACCTG 27 gene (uidA, GUS) from E. coli encodes beta-glucuronidase GGAGAAACTGCATCAGCCGATTATCATCACCGAATACG 28 Glycine max (soy) Lectin gene of the Le1 family, encodes a lectin GTTCTCATTACCTATGATGCCTCCACCAGCCTCTTTG 29th Zein gene of the zein family from Zea Mays (corn), encodes zein TGCGGCGAGCGTCTTACAACAACCAATTGC thirty the bar gene from Streptomyces hygroscopicus, encodes TCTACACCCACCTGCTGAAGTCCCTGGAGGCA phosphotricin acetyltransferase 31 cry1Ab gene from Bacillus thuringiensis encodes a Bt toxin CGTCAACGTGCCCGGTACTGGTTCCCTCTG 32 EPSPS gene from (Agrobacterium tumefaciens CP4), encodes 5-enolpyruvyl shikimate-3-phosphate synthase CAAGTCGATCTCCCACCGGTCCTTCATGTTCG 33 gene (pat1) from Solarium tuberosum (potato), encodes a patatin precursor GCAGCAAGTTCTTACATGACTGATTATTACCTTTCTACTGTTTTTTCAAG

Synthesis of probes and primers

Oligonucleotides are synthesized on an automatic synthesizer (for example, Pharmacia's Gene Assembler model) using the standard amidophosphite method. Differentiating oligonucleotides contain a 5'- or 3'-terminal group, ensuring their immobilization on the surface of the chip, for example, a 5'-terminal phosphate group, which is introduced by chemical phosphorylation during synthesis. One of a pair of specific oligonucleotides for PCR is modified at the 5'-end with a label that is selected from the group consisting of: catalytic, ligand, fluorescent and radioactive, which is introduced by chemical methods or enzymatically.

DNA isolation and preparation of material for amplification

Isolation of DNA is carried out from a biological sample containing GMI, included in the group consisting of fruits or juice of plants, grain, flour or food products, which include genetically modified plants.

DNA isolation is carried out using conventional methods based, for example, on alkaline lysis, the use of detergents, proteinase K, extraction with phenol, chloroform and / or a phenol / chloroform mixture, or purification on diatomaceous earth or using silica.

Positive and negative controls

In the framework of this invention, a positive control is used in the biochip and in the diagnostic kit to confirm compliance with the optimal conditions for the procedures necessary to identify the declared transgenic plants, namely hybridization and detection. For this purpose, the differentiating oligonucleotide SEQ ID NO: 34 is applied to the chip, to which the oligonucleotide SEQ ID NO: 35 is complementary. The positive control oligonucleotide is added to the sample after PCR or processing with phage lambda exonuclease and hybridization is carried out under the same conditions as for the determined markers.

Negative controls in the framework of this invention are used to confirm compliance with optimal hybridization conditions. As a negative control, an oligonucleotide, partially complementary to the positive control DNA sequence, or an oligonucleotide, partially complementary to any of the specific PCR primers that are modified at the 5'-end with the label from the group given in p. “Synthesis of probes and primers, is immobilized onto a chip” ". In the sequence of the differentiating oligonucleotide serving as a negative control, the number and position of nucleotides that do not match the DNA sequence of the positive control or specific oligonucleotides for PCR are selected so that this differentiating oligonucleotide is able to form and maintain an imperfect duplex with a single stranded DNA of a positive control or specific oligonucleotide for PCR if hybridization and washing conditions are not observed, namely when hybridization and washing under conditions milder than optimal. As an example, which includes but is not limited to the scope of the invention, a differentiating oligonucleotide ID SEQ NO: 36 is shown in the sequence listing, which is immobilized on the surface of the chip and used as a negative control.

Tag selection

A large number of patents are known in which various types of tags are considered for constructing biological chips. Known classification of labels into groups consisting of: catalytic, ligand, fluoresceit or radioactive molecules [9]. These patents are incorporated by reference. The catalytic molecule is selected from the group consisting of hemin, cyanocobalamin or flavin. The ligand molecule is selected from the group consisting of biotin, dioxigenin or dinitrobenzene. The fluorescent molecule is selected from the group consisting of FAM, TAMRA, Cy3, Cy5, Cy7, R6G, R110, ROX or JOE.

Amplification of DNA by PCR

Single-stranded DNA fragments of typical GMI sequences shown in Table 1 are obtained by symmetric PCR using highly specific primers.

PCR conditions

The PCR mixture contains 1x reaction buffer, for example, from 10 to 80 mM Tris-HCl pH 8.8, up to 50 mM KCl and / or up to 25 mM (NH ₄ ) ₂ SO ₄ , and / or 0, 1% Triton X-100, and / or 0.1% Tween 20, and / or 0.8% Nonidet P40, from 1 mm to 5 mm, optimally from 1.5 mm to 2.5 mm, MgCl ₂ or MgSO ₄ , from 10 μg to 300 μg, optimally from 50 μg to 200 μg, the DNA of the analyzed sample and at least one pair of specific oligonucleotide primers for each marker. The total number of pairs of specific oligonucleotides can vary from 1 to 11 in combinations to identify at least one GMI marker. The composition of the PCR mixture includes from 1 unit. up to 20 units, optimally from 3 units. up to 7 units, most optimally from 4 units. up to 6 units Taq polymerase activity. The composition of the PCR mixture may include reagents that increase the specificity and effectiveness of PCR, for example, such as betaine, ectain and its derivatives, as well as trehalose, dimethyl sulfoxide, glycerol.

The concentration of oligonucleotides in PCR can be from 0.05 μm to 0.3 μm, optimally from 0.1 μm to 0.25 μm.

The volume of the reaction mixture can be from 5 μl to 200 μl, optimally from 20 μl to 50 μl. The reaction can be carried out with or without mineral oil, depending on the design of the DNA amplifier. PCR is carried out using a DNA amplifier (for example, "Terzik", DNA technology, Russia or "Mastercycler", Eppendorf, Germany) under the conditions shown in table 4.

Table 4 PCR conditions Program step Temperature ° C Incubation time, sec The number of cycles one 94 ± 2 180 ± 120 one 2 94 ± 2 15 ± 10 from 35 to 45 3 61 ± 3 15 ± 10 four 72 ± 2 20 ± 10 5 72 ± 2 from 180 to 300 one

PCR treatment of products with phage lambda exonuclease

One of a pair of PCR primers contains a phosphate group at the 5'-end. This is necessary in order for the chain of the DNA fragment that is synthesized during PCR with this primer to be efficiently hydrolyzed by the phage lambda exonuclease. Since the second of the primer pairs at the 5 ′ end contains biotin or a fluorescent label, this DNA strand is protected from degradation by exonuclease. This makes it possible to obtain single-stranded DNA labeled at the 5'-end with biotin or a fluorescent label after processing PCR products with an exonuclease of phage lambda. Hybridization with single-stranded DNA is more efficient, which improves the sensitivity and reproducibility of the diagnosis. To obtain single-stranded DNA, asymmetric PCR is often used. The method of obtaining single-stranded DNA using asymmetric PCR requires a large number of PCR cycles and up to a 50-fold excess of one of the primers, which leads to an increase in PCR time and increases the cost of the reaction. The use of phage lambda exonuclease to obtain single-stranded DNA allows the use of standard symmetric PCR, which is more efficient and economical both in terms of the number of reagents and the time taken. From 5 to 20 units of phon lambda phonon exonuclease activity are used to hydrolyze PCR products. Processing time is from 30 to 90 minutes. More preferably, 10 units of the enzyme are used and the reaction is carried out for 60 minutes at a temperature of 37 ° C.

Hybridization

Labeled PCR products hybridize with DNA probes (differentiating oligonucleotides) immobilized on a biochip in a specially selected buffer.

Hybridization Conditions

Hybridization is carried out in a buffer containing 0.5 × -5 × SSC, 0.1% SDS, up to 25% formamide and / or up to 10 mm EDTA from 30 minutes to 1 hour at 35-50 ° C. Then the chip is washed at room temperature on an orbital shaker from 5 to 10 times with solutions of 0.5 × -5 × SSC, 0.1% SDS. In the case of using enzymes for the manifestation of hybridization results, the last three washes are carried out with an SDS-free SSC solution.

Peroxidase reaction

If the PCR products are labeled with a biotype, for further identification it is necessary to conduct a reaction with a conjugate of streptavidin-peroxidase or streptavidin-phosphatase.

Biochip structure

The biochip for identification of GMI is a supporting element on the surface of which more than 6 clusters with differentiating oligonucleotides selected from SEQ ID NO: 23-33, at least one differentiating positive control oligonucleotide shown in SEQ ID NO: 34 are immobilized, and, at least one negative control oligonucleotide shown in SEQ ID NO: 36. Differentiating oligonucleotides are applied in a cluster in four or nine repetitions. The number of clusters located in the working area of each individual chip can be 15. Figure 2 shows one of the variants of the structural diagram of the biochip, which includes, but does not limit the possibility of using other options, for example, presented in Figure 1 with three positive controls.

The preferred form of the multichip is a flat rectangular element made of a solid or flexible material of different thicknesses. The thickness of the carrier may be from 0.05 mm to 5 mm. The supporting element of the chip is made of materials included in the group consisting of: polymers, glass, metals, ceramics, or combinations thereof. The polymers are selected from the group consisting of: polymethyl methacrylate, polybutyl methacrylate, polyvinyl chloride, polycarbonate, copolymers of methyl methacrylate and / or copolymers of butyl methacrylate with other monomers such as styrene, acrylonitrile.

The surface of the supporting element can be modified as needed or coated with some material capable of immobilizing oligonucleotides.

In one embodiment of the biochip, which includes, but does not limit the scope of the invention, the arrangement of the positive control clusters is such that the positive control clusters are located in at least two diagonally located corners of the working area, thus indicating the boundaries of the working area, which simplifies the procedure for identifying the location of the working area when scanning diagnostic results. Figure 1 shows the hybridization pattern obtained by scanning the biochip in the diagnosis of a food product, which shows an example of the placement of three positive controls in the corners of the working area of the biochip (section "data interpretation").

Multi-chip identification

The biochip identifier may include a batch number code and / or production date, information about the list of placed differentiating oligonucleotides. The identifier can be made in the form of a barcode or an element with magnetic recording of information.

The use of a unique biochip code will provide a quick search of information in the databases of all samples during parallel screening of GMOs.

Interpretation of analysis data

The signal intensity of the resulting perfect or imperfect duplexes within each group of clusters is significantly higher than the signal intensity of the negative controls, which allows reliable identification of GMI. Detection and / or identification of GMI is carried out by comparative analysis of the signal level of the test sample, positive and negative controls. Interpretation of the results is carried out on the basis of a hybridization picture, which is obtained using an experimental setup, including, for example, a scanner, a computer, and special software. Figure 1 shows the hybridization pattern with colorimetric labels obtained by scanning the biochip, to determine the possible content of GMI in a food sample isolated from 0.1% soy.

In this version of the biochip, clusters of 1, 2, 3 positive controls with a maximum signal were identified at the three corners of the working area of the biochip. In the second horizontal row there is a cluster 4 corresponding to the marker S35, and a cluster 5 corresponding to the marker Nos. In the third horizontal row is cluster 6, corresponding to the Lectin marker.

The interpretation of the results is as follows. The intensities of fluorescent or colorimetric signals in each group of DNA probes containing oligonucleotides that are species-specific for one of the 11 GMI markers are compared using special software with the groups of probes of the positive (maximum signal intensity) and negative (minimum signal intensity) control. As a result, a hybridization pattern is formed that is different for the groups of positive control, negative control and for clusters that characterize GMI markers, which allows one to unambiguously interpret the obtained data.

Diagnostic kit

Another object of the invention is a kit for identifying GMI markers, including:

a) at least one biochip for a one-time diagnosis, containing more than 6 differentiating oligonucleotides specific for identifiable markers of transgenic plants, with the sequences shown in SEQ ID NO: 23-33, one or more differentiating oligonucleotides as a positive control, with the sequence presented in SEQ ID NO: 34, and one or more differentiating oligonucleotides as a negative control, with the sequence represented in SEQ ID NO: 36;

b) specific oligonucleotides used as PCR primers to identify GMI markers whose sequences are presented in SEQ ID NO: 1-22, and a specific oligonucleotide shown in SEQ ID NO: 35, which is complementary to the differentiating oligonucleotide of the positive control SEQ ID NO: 34 placed on a biochip in clusters of positive control;

c) reagents that are selected from the group consisting of: reagents for sample preparation, reagents for conducting multi-primer symmetric PCR, reagents for hybridization, or combinations thereof and an exonuclease of phage lambda.

Examples

The following are examples that include, but are not limited to, the scope of the invention.

Example 1. Preparation of the surface of the biochip (surface modification)

A matrix containing amino groups with a density of 1-2 groups / nm ² is prepared using a solution of 3-aminopropyltriethoxysilane (APTES) in various solvents depending on the type of substrate. For example, glass substrates were incubated for one hour in a solution of 3-aminopropyltriethoxysilane in acetone or in ethanol. APTES concentration varied from 0.1 to 10% by volume. If the reaction is carried out in acetone, the glasses are washed three times with acetone for 5 minutes, then ethanol 2 times for 3 minutes and dried at a temperature of 120-130 ° C for 20 minutes.

Example 2. Sewing oligonucleotide probes to the surface of amino-modified glass

Probing

A reaction mixture containing 1 μM oligonucleotide and 60 mM 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride in 0.1 M 1-methylimidazole, pH 7, was applied to the matrix surface with a robot (Xpress Lane, USA). The droplet volume of each probe is at least 0.005 μl. Matrices coated with oligonucleotides were placed in a sealed container, kept at room temperature for 1 hour in a humid atmosphere and washed on a shaker with a 0.5 mM NaCl solution containing 0.1% SDS for 10 minutes, then with distilled water. The biochip was dried and stored at a temperature of 4 to 8 ° C. The concentration of oligonucleotide in the volume of drops of each probe is from 0.5 to 100 μmol / L.

As probes, differentiating oligonucleotides selected from ID SEQ No: 23-33 were used. Oligonucleotides were applied in clusters of 4 points.

Example 3. Carrying out multi-primer symmetric PCR for hybridization of the biochip with seven clusters to identify markers Zein, Lectin, NOS, 35S, OCS, NPTII, GUS

The PCR mixture consisted of 10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.8% Nonidet P40, 2 mM MgCl ₂ , 100 ng of DNA of the analyzed sample, specific oligonucleotide primers ID SEQ NO: 1, 3, 5, 7, 9, 11, 13 and primers ID SEQ NO: 2, 4, 6, 8, 10, 12, 14, modified with biotin at the 5'-position, are used in pairs at concentrations: ID SEQ NO: 1 and ID SEQ NO: 2 at a concentration of 0.25 μM; ID SEQ NO: 3 and ID SEQ NO: 4 at a concentration of 0.25 μM; ID SEQ NO: 5 and ID SEQ NO: 6 at a concentration of 0.1 μM; ID SEQ NO: 7 and ID SEQ NO: 8 at a concentration of 0.1 μM; ID SEQ NO: 9 and ID SEQ NO: 10 at a concentration of 0.1 μM; ID SEQ NO: 11 and ID SEQ NO: 12 at a concentration of 0.15 μM; ID SEQ NO: 13 and ID SEQ NO: 14 at a concentration of 0.15 μM, and 5 units. Taq polymerase in a volume of 20 μl. The reaction was carried out on a Mastercycler DNA amplifier (Eppendorf, Germany) under the conditions shown in table 5.

Table 5 PCR conditions Program step Temperature ° C Incubation time, sec The number of cycles one 94 180 one 2 94 fifteen 39 3 61 fifteen four 72 twenty 5 72 180 one

Example 4. Carrying out multi-primer symmetric PCR for hybridization of the biochip and identification of markers: bar, cry1Ab, EPSPS, Patatin

The PCR mixture consisted of 10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.8% Nonidet P40, 2 mM MgCl ₂ , 100 ng of DNA of the analyzed sample, specific oligonucleotide primers ID SEQ NO: 15, 17, 19, 21 and primers ID SEQ NO: 16, 18, 20, 22. The oligonucleotides shown in SEQ ID NO: 15, 17, 19, 21 contained a 5'-terminal phosphate. The oligonucleotides shown in SEQ ID NO: 16, 18, 20, 22 contained a Cy5 fluorescent label at the 5 ′ end. Primers are used in pairs at concentrations of: ID SEQ NO: 15 and ID SEQ NO: 16 at a concentration of 0.25 μM; ID SEQ NO: 17 and ID SEQ NO: 18 at a concentration of 0.25 μM; ID SEQ NO: 19 and ID SEQ NO: 20 at a concentration of 0.25 μM; ID SEQ NO: 21 and ID SEQ NO: 22 at a concentration of 0.25 μM and 5 units. Taq polymerase in a volume of 20 μl. The reaction was carried out on a Mastercycler DNA amplifier (Eppendorf, Germany) under the conditions shown in table 5.

Example 5. Hybridization

To 20 μl of the PCR mixture, 5 μl of 20x SSC and 2.5 μl of 10% SDS were added, applied to the working area of the substrate, covered with a 16 × 21 mm film and incubated for 1 hour at 45 ° C. At the end of the hybridization reaction, the surface of the chip was washed on an orbital shaker with the following solutions:

A) 2 × SSC, 0.1% SDS - 2 times for 5 minutes;

B) 0.2 × SSC, 0.1% SDS - 2 times for 5 minutes;

B) 0.2 × SSC - 3 times in 1 minute.

After washing, the biochip was dried at room temperature.

Example 6. The peroxidase reaction

The initial streptavidin-peroxidase conjugate (Imbio, Russia) was diluted 200 times with 1x SSC containing 1% BSA. The conjugate was applied to the working area of the substrate, covered with a film and kept in a humid atmosphere at room temperature for 30 minutes. The unbound conjugate was washed with 2x SSC for 5 minutes, 1x SSC for 5 minutes and 0.1 × SSC for 1 minute.

The substrate was poured with a freshly prepared solution of the substrate dimethylaminobenzidine. For this, no more than half an hour before the reaction, 1 mg DAB was dissolved in 1 ml of 0.1 × SSC, 20 μl of a 3% hydrogen peroxide solution was added and mixed. The working area was poured with the prepared substrate solution, covered with a film and kept for 10 to 30 minutes at room temperature. In the case of a positive reaction, brown spots of the oxidized substrate appeared. Then the glass was washed with distilled water and placed in a vertical position in a container for drying and further storage.

Example 7. Probe Detection

To obtain images of hybridization microzones, a Nikon CoolScan 9000ED slide scanner was used. Then carried out a quantitative image processing using a computer program.

Figure 1 presents a picture of the hybridization of DNA samples. It can be seen that each identified DNA has its own individual hybridization pattern. Clusters corresponding to the negative control did not appear. Positive controls are located in the three corners of the working area of the biochip and determine the boundaries of the working area during quantitative image processing.

To obtain images of clusters using fluorescent labels, a fluorescence chip detector manufactured by the company Molecular Medical Technologies was used. Figure 5 presents the image of the result of the multi-primer symmetric PCR and hybridization of the biochip when identifying markers: bar, cry1Ab, EPSPS, Patatin.

Example 8. Checking the effectiveness of hybridization after processing PCR products with an exonuclease phage lambda

Got 3 identical PCR mixture in a volume of 20 μl each. Composition of the PCR mixture: 67 mM Tris-HCl, pH 8.8; 16 mM (NH ₄ ) ₂ SO ₄ ; 2 mM MgCl ₂ ; 0.2 mM dNTP; 0.01% Tween 20, 0.25 μM of each oligoyucleotide, which are presented in SEQ ID No. 1-14. The oligonucleotides shown in SEQ ID Nos. 1, 3, 5, 7, 9, 11, and 13 contained a 5'-terminal phosphate. The oligonucleotides shown in SEQ ID Nos. 2, 4, 6, 8, 10, 12, and 14 contained a Cy5 fluorescent label at the 5 ′ end, a mixture of recombinant plasmid DNAs containing cloned fragments of 35S, NOS, OCS, NPTII, GUS, LECTIN and ZEIN, in an amount of 0.05 ng each and 5 units. Taq polymerase activity. PCR conditions are shown in table 6.

Table 6 Program step Temperature ° C Incubation time, sec The number of cycles one 94 180 one 2 94 fifteen 38 3 61 fifteen four 72 twenty 5 72 180 one

After PCR, 1 μl of phage lambda phonon exonuclease preparation containing 10 activity units was added to sample 1 and incubated for 1 hour at 37 ° C. The reaction was stopped by adding 2 μl of a stop solution (composition of a stop solution: 100 mM EDTA and 0.5 μM oligopucleotide shown in SEQ ID NO: 35 with a fluorescent label Cy5 at the 5'-end) and the sample was incubated for 10 min at 80 ° FROM. A stop solution was added to samples 2 and 3. Then, sample 2 for DNA denaturation was incubated in a boiling bath for 5 min, and then placed in ice. Sample 3 was not subjected to any treatment before hybridization, i.e. served as a control of the efficacy of exonuclease use and thermal denaturation of DNA.

After that, hybridization was performed. For this, 2.5 μl of 20 × SSC and 2.5 μl of 1% SDS were added to each microtube with samples 1, 2, and 3 and mixed by piloting. The resulting mixture was applied to the middle of the working area of the microchip (a separate microchip was used for each sample), covered with a 16 × 21 mm film, the microchips were placed in a humid chamber and incubated for 1 hour at a temperature of 45 ° С in an air thermostat. At the end of the reaction, the film was removed from each microchip and the washing was performed on the orbital shaker with the following solutions (the temperature of the solutions was 28 ° C):

2 × SSC, 0.1% SDS 2 times for 5 minutes 0.2 × SSC, 0.1% SDS 2 times for 5 minutes 0.2 × SSC 3 times in 1 minute

After washing, the microchips were dried at room temperature for 15 minutes and the results were detected using a VAF-1 chip detector. The results are shown in Figure 4: a) for sample 1, b) for sample 2, and c) for sample 3. The highest signal intensity in the clusters of the analyzed genetic markers was observed after processing sample 1 with an exonuclease. For sample 2 subjected to thermal denaturation, maximum fluorescence saturation was not observed, whereas in control sample 3, a positive hybridization signal was observed only in clusters of positive and negative controls. This example demonstrates that the use of exonuclease significantly increases the effectiveness of the analysis.

Industrial applicability

The method for identifying GMOs on a biochip compares favorably with the speed of analysis, the simplicity of the method used to prepare a DNA sample for hybridization, the ability to simultaneously identify up to 11 of the most common GMI markers, the possibility of analysis in laboratory or field conditions, and also relative cheapness. The texts of patents and patent applications are considered in the text of this description as references.

Literature

1. Lipp M.I. et al. Decision of the European Parliament and Council No. 1829/2003 of 09/22/2003 "On Genetically Modified Food and Feed." Journal of AOAS Intern., Vol. 82, No. 4, pp. 923-928 (1999).

2. Park H. et al. Detection method of genetic recombinant food and detection kit of that. W02001098523 (12/27/2001).

3. Lauter F.R. et al. Test kit and method for quantitatively detecting genetically modified DNA in foodstuff by means of fluorescence-coupled PCR. US Patent Apll. 20030148278 (August 7, 2003).

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Claims (18)

1. A method for identifying markers of genetically modified plants, including analysis of a sample with the studied DNA using direct and reverse primers specific for genes of GMO markers (genetically modified sources), and a biochip, conducting multi-primer PCR in the sample, processing the products of the PCR reaction with an enzyme, incubating PCR product with differentiating oligonucleotides immobilized on the surface of the biochip under hybridization conditions, determination of the presence of GMI markers by detection of the formed hybridization complexes on the biochip, characterized in that the GMI markers identified in Table 1 are identified, the biochip contains more than 6 types of clusters of differentiating oligonucleotides selected from the sequences shown in SEQ ID NO: 23-33, with primers being used as specific primers the sequences of which are selected from the group consisting of SEQ ID NO: 1-22, as PCR, use a multi-primer symmetric PCR, the products of which are preferably treated in a volume of 20 μl, preferably 1 μl of exonuclease f and lambda in a concentration of 5 to 20 activity units per microliter is incubated for 30-90 min, preferably at a temperature of 37 ° C, where in a pair of forward and reverse primers, one primer contains a detectable label that is used to identify GMO markers, the differentiating oligonucleotide SEQ ID NO: 36, partially complementary to the oligonucleotide SEQ ID NO: 8, is used as a negative control, and the differentiating oligonucleotide SEQ ID NO: 34, which is specifically complementary, is used oligonucleotide of SEQ ID NO: 35. 2. The method according to claim 1, characterized in that the test DNA is isolated from at least one sample containing GMI, which is included in the group consisting of fruits or juice of plants, grain, flour or food products, which include genetically modified plants. 3. The method according to claim 1, characterized in that the defined label is selected from the group consisting of catalytic, ligand, fluorescent or radioactive molecules, the catalytic molecule being selected from the group comprising hemip, cyanocobalamin or flavin, the ligand molecule is selected from the group including biotin, dioxigenin or dinitrobepzole, and the fluorescent molecule is selected from the group consisting of FAM, TAMRA, Cy3, Cy5, Cy7, R6G, R110, ROX or JOE. 4. The method according to claim 1, characterized in that the detection and / or identification of GMI is carried out by comparative analysis of the signal level of the test sample, positive and negative controls. 5. A specific oligonucleotide as a primer with a symmetric PCR multiprimer for a method for identifying genetically modified plant markers according to claims 1-4, which is characterized by a nucleotide sequence selected from the sequences shown in SEQ ID NO: 4, 7, 8, 11-14, 21 , 22. 6. Differentiating oligonucleotide as a probe for identifying genetically modified plant markers for use in the GMI identification method according to claims 1-4, which is characterized by a nucleotide sequence selected from the sequences shown in SEQ ID NO: 24, 28, 29, 33. 7. A biochip for identifying GMO markers, which is a supporting element on which differentiating oligonucleotides are immobilized in the form of probes forming clusters, characterized in that more than 6 types of clusters with differentiating oligonucleotides selected from SEQ ID NO: 23-33 are immobilized on its surface wherein the biochip contains at least one cluster comprising a positive control oligonucleotide shown in SEQ ID NO: 34, and at least one cluster comprising a negative control oligonucleotide To presented in SEQ ID NO: 36. 8. The biochip according to claim 7, characterized in that the supporting element of the chip is made of materials included in the group consisting of polymers, glass, metals, ceramics, or combinations thereof. 9. The biochip of claim 8, wherein the polymers are selected from the group consisting of polymethyl methacrylate, polybutyl methacrylate, polyvinyl chloride, polycarbonate, copolymers of methyl methacrylate and / or copolymers of butyl methacrylate with other monomers such as styrene, acrylonitrile. 10. The biochip according to claim 7, characterized in that the supporting element of the chip is made in the form of flat platforms, films with a thickness of 0.05 to 5.0 mm. 11. The biochip according to claim 7, characterized in that it further comprises an identifier made in the form of a barcode or element with magnetic recording. 12. The biochip according to claim 11, characterized in that the biochip identifier includes a batch number code and / or production date and information about the list of placed differentiating oligonucleotides. 13. A kit for identifying GMI markers, including:
a) at least one biochip for a one-time diagnosis, containing more than 6 types of differentiating oligonucleotides specific for identifiable markers of transgenic plants, with the sequences shown in SEQ ID NO: 23-33, one or more differentiating oligonucleotides as a positive control, with the sequence shown in SEQ ID NO: 34, and one or more differentiating oligonucleotides as a negative control, with the sequence shown in SEQ ID NO: 36,
b) reagents that are selected from reagents for sample preparation, reagents for conducting multi-primer symmetric PCR, reagents for hybridization, and combinations thereof and an exonuclease enzyme,
c) specific oligonucleotides used as PCR primers to identify GMI markers whose sequences are presented in SEQ ID NO: 1-22, and a specific oligonucleotide shown in SEQ ID NO: 35, which is complementary to the differentiating positive control oligonucleotide SEQ ID NO: 34 placed on the biochip in clusters of positive control. 14. The combination of oligonucleotides for detecting the NOS marker, mainly for identifying the 3 'untranslated region of the nopaline synthase gene, characterized in that it includes oligonucleotides with the sequences SEQ ID NO: 24 and SEQ ID NO: 3 and SEQ ID NO: 4. 15. The combination of oligonucleotides to identify the NPTII marker, mainly for identifying a gene encoding neomycin phosphotransferase II, characterized in that it comprises oligonucleotides with the sequences SEQ ID NO: 26 and SEQ ID NO: 7 and SEQ ID NO: 8. 16. The combination of oligonucleotides to identify the Lectin marker, mainly for the identification of the Le1 family gene from Glycine max, characterized in that it comprises oligonucleotides with the sequences SEQ ID NO: 28 as a probe and two specific SEQ ID NO: 11 and SEQ ID NO: 12. 17. The combination of oligonucleotides for detecting the Zein marker, mainly for identifying a gene of the zein family from Zea Mays, characterized in that it comprises oligonucleotides with the sequences SEQ ID NO: 29 and SEQ ID NO: 13 and SEQ ID NO: 14. 18. The combination of oligonucleotides to identify the Patatin marker, mainly for the identification of the pat1 gene from Solanum tuberosum encoding a patatin precursor, characterized in that it comprises oligonucleotides with the sequences SEQ ID NO: 33 and SEQ ID NO: 21 and SEQ ID NO: 22.

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