MX2007003725A - Corn event das-59122-7 and methods for detection thereof - Google Patents

Abstract

The invention provides DNA compositions that relate to transgenic insect resistant maize plants. Also provided are assays for detecting the presence of the maize DAS-59122-7 event based on the DNA sequence of the recombinant construct inserted into the maize genome and the DNA sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.

Description

MAIZE EVENT DAS-59122-7 AND METHODS OF DETECTING THE SAME FIELD OF THE INVENTION Embodiments of the present invention relate to the field of plant molecular biology, more specifically an embodiment of the invention relates to a DNA construct for conferring insect resistance to plants. Embodiments of the invention relate more specifically to an insect resistant DAS-59122-7 maize plant and to tests for the presence of DNA from DAS-59122-7 maize plants in a sample and compositions therewith. BACKGROUND OF THE INVENTION An embodiment of this invention relates to a maize plant (Zea mays) DAS-59122-7 resistant to insects, also called corn line DAS-59122-7 or corn event DAS-59122-7 and with the DNA of a plant expression construct of corn plants DAS-59122-7 and the detection of the transgene / flanking insertion region in maize plants DAS-59122-7 and with the progeny thereof. Maize is an important crop and is a primary food source in many areas of the world. The damage caused by insect pests is one of the main factors in the loss of corn crops worldwide, despite the use of protective measures such as chemical pesticides. In view of this, insect resistance in crops, such as corn, has been genetically engineered to control the damage caused by insects and reduce the need for traditional chemical pesticides. A group of genes that has been used to produce insect-resistant transgenic crops are the delta-endotoxins of Bacillus thuringiensis (B.t.). Das delta-endotoxins have been successfully expressed in crop plants, such as cotton, potatoes, rice, sunflower, as well as corn, and have demonstrated excellent control of insect pests. (Perlak, FJ et al (1990) Bio / Technology 8, 939-943; Perlak, FJ et al. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al. (1993) Bio / Technology 11: 1151-1155; Tu et al. (2000) Nature Biotechnology 18: 1101-1104; PCT Publication No. WO 01/13731; and Bing J et al. (2000) Efficacy of CrylF Transgenic Maize, 14th Biennial International Plant Resistance to Insects Workshop, Fort Collins, CO). It is known that the expression of foreign genes in plants is affected by their location in the plant genome, perhaps due to the structure of chromatin (eg, heterochromatin) or the proximity of regulatory elements of transcription (eg, enhancers) near of the site of integration (Weising et al., Ann. Rev. Genet 22: 421-477, 1988). At the same time, the presence of the transgene in different locations in the genome will affect the general phenotype of the plant in different ways. For this reason, it is often necessary to examine a large number of events in order to identify an event characterized by an optimal expression of the introduced gene of interest. For example, it has been observed in plants and other organisms that there may be a great variation in the expression levels of a gene introduced between the different events. There may also be differences in spatial or temporal expression patterns, for example, differences in the relative expression of a transgene in different plant tissues, which may not correspond to the expected patterns of the transcriptional regulatory elements present in the introduced genetic construct. . For this reason, it is common to produce hundreds to thousands of different events and examine those events for a single event that presents the desired levels and expression patterns of the transgene for commercial purposes. An event with the desired levels or expression patterns of the transgene is useful to introduce the transgene into other genetic antecedents by sexual crossing using conventional breeding methods. The progeny of said crosses retains the expression characteristics of the transgene of the original transformant. This strategy is used to ensure reliable gene expression in numerous varieties that are well adapted to local growth conditions. It would be advantageous to be able to detect the presence of a particular event in order to determine if the progeny of a sexual cross contains a transgene of interest. In addition, a method to detect a particular event would be very helpful in complying with pre-commercialization and labeling approval standards of foods derived from recombinant crop plants, for example, or for use in environmental monitoring, trait monitoring in crops in the field or monitoring of the products derived from a crop crop, as well as its use to ensure compliance of the parties subject to regulatory or contractual terms. It is possible to detect the presence of a transgene using any nucleic acid detection method known in the art including, for example, polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods are generally based on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructions, the coding region is interchangeable. As a result of this, it is possible that such methods are not used to distinguish different events, in particular those that are produced using the same DNA construct or very similar constructions unless the DNA sequence of the flanking DNA adjacent to the inserted heterologous DNA is known. For example, a PCR assay specific to the event is described in U.S. Pat. N °: 6,395,485 for the detection of the elite event GAT-ZM1. Therefore, it would be convenient to have a simple and distinctive method to identify event DAS-59122-7. BRIEF DESCRIPTION OF THE INVENTION Embodiments of this invention relate to methods for producing and selecting an insect-resistant monocotyledonous cultivation plant. More specifically, a DNA construct is provided which, when expressed in plant cells and plants confers insect resistance. According to one aspect of the invention, a DNA construct capable of introducing and replicating in a host cell is provided which, when expressed in plant and plant cells confers insect resistance to said plant cells and plants. The DNA construct is composed of a DNA molecule called PHI17662A and includes three (3) transgenic cassette expression cassettes. The first expression cassette comprises a DNA molecule that includes the promoter, a 5 'non-translated exon and the first intron of the gene of ubiquitin (Ubi-1) of corn (Christensen et al. (1992) Plant Mol. Biol. 18: 675-689 and Christensen and Quail (1996) Transgenics Res. 5: 213-218) operatively linked to a DNA molecule encoding a Bt d-endotoxin, identified as Cry34Abl (U.S. Patent Nos .: 6,127,180, 6,624,145 and 6,340,593), operatively linked to a DNA molecule comprising Pin II of the terminator of the isolated transcription of potato (Gyheung An et al. (1989) Plant Cell 1: 115-122). The second cassette of transgenic expression of the DNA construct comprises the DNA molecule encoding the wheat peroxidase promoter (Hertig et al (1991) Plant Mol. Biol. 16: 171-174) operably linked to a DNA molecule which codes for a Bt d-endotoxin identified as Cry35Abl (U.S. Patent Nos .: 6,083,499, 6,548,291 and 6,340,593) operatively linked to a DNA molecule comprising a Pin II transcription terminator isolated from potato (Gyheung An et al. (1989) Plant Cell 1: 115-122). The third transgenic expression cassette of the DNA construct comprises a cauliflower mosaic virus (CaMV) DNA molecule (Odell JT et al (1985) Nature 313: 810-812; Mitsuhara et al. (1996) Plant Cell Physiol., 37: 49-59) operably linked to a DNA molecule encoding a phosphinothricin acetyltransferase (PAT) gene (Ohlleben W. et al. (1988) Gene 70: 25-37) operably linked to a DNA molecule which comprises the transcription terminator 3 '35S (CaMV) (see, Mitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59). Plants containing the DNA construct are also provided. According to another embodiment of the invention, compositions and methods are provided to identify a new maize plant designated DAS-59122-7, wherein said methods are based on primers or probes that specifically recognize the 5 'and / or 3' flanking sequence. of DAS-59122-7. DNA molecules are provided which comprise primer sequences which, when used in a PCR reaction, will produce unique amplicons for the transgenic event DAS-59122-7. These molecules can be selected from the group consisting of: 5 '-GTGGCTCCTTCAACGTTGCGGTTCTGTC-3' (SEQ ID NO: 1); 5 '-CGTGCAAGCGCTCAATTCGCCCTATAGTG-3' (SEQ ID NO: 2); 5 '-AATTGAGCGCTTGCACGTTT-3' (SEQ ID N °: 3); 5 '-AACAACAAGACCGGCCACACCCTC-3' (SEQ ID N °: 4); 5'-GAGGTGGTCTGGATGGTGTAGGTCA-3 '(SEQ ID NO: 5); 5 '-TACAACCTCAAGTGGTTCCTCTTCCCGA-3' (SEQ ID N °: 6); 5'-GAGGTCTGGATCTGCATGATGCGGA-3 '(SEQ ID N °: 7); 5 '-AACCCTTAGTATGTATTTGTATT-3' (SEQ ID N °: 8); 5 '-CTCCTTCAACGTTGCGGTTCTGTCAG-3' (SEQ ID NO: 9); 5 '-TTTTGCAAAGCGAACGATTCAGATG-3' (SEQ ID N0: 10); 5'-GCGGGACAAGCCGTTTTACGTTT-3 '(SEQ ID NO: 11); 5 '-GACGGGTGATTTATTTGATCTGCAC-3' (SEQ ID N °: 12); '-CATCTGAATCGTTCGCTTTGCAAAA-3' (SEQ ID NO: 13); 5 '-CTACGTTCCAATGGAGCTCGACTGTC-3' (SEQ ID NO: 14); 5'-GGTCAAGTGGACACTTGGTCACTCA-3 '(SEQ ID NO: 15); 5'-GAGTGAAGAGATAAGCAAGTCAAAG-3 '(SEQ ID N °: 16); 5 '-CATGTATACGTAAGTTTGGTGCTGG- 3' (SEQ ID NO: 17); 5 '-AATCCACAAGATTGGAGCAAACGAC- 3' (SEQ ID N °: 18) 5 '-CGTATTACAATCGTACGCAATTCAG- 3' (SEQ ID N °: 36); 5 '-GGATAAACAAACGGGACCATAGAAG- 3' (SEQ ID N °: 37) and complements thereof. The plant and seed comprises these molecules constitute an embodiment of this invention. In addition, sets of elements are provided that employ these primer sequences for the identification of event DAS-59122-7. A further embodiment of the invention relates to the specific flanking sequences of DAS-59122-7 described herein, which can be used to develop specific identification methods for DAS-59122-7 in biological samples. More particularly, the invention relates to the 5 'and / or 3' flanking regions of DAS-59122-7, SEQ ID N °: 19 for flanking region 5 'and SEQ ID N °: 20 for flanking region 3'. , respectively, that can be used to develop specific primers and probes. A further embodiment of the invention relates to methods for identifying the presence of DAS-59122-7 in biological samples based on the use of said specific primers or probes. According to another embodiment of the invention, methods are provided for detecting the presence of the DNA corresponding to the maize event DAS-59122-7 in a sample. Said methods comprise: (a) putting the DNA comprising sample in contact with a set of DNA primers which, when used in a nucleic acid amplification reaction with genomic DNA extracted from the corn event DAS-59122-7, produces an amplicon that is diagnostic for the corn event DAS-59122-7; (b) carrying out a nucleic acid amplification reaction, whereby an amplicon is produced; and (c) detecting said amplicon. DNA molecules comprising the new transgene / flanking insertion region, SEQ ID NO: 21, 5 'flanking region plus 1000 internal and SEQ ID NO: 22, 3' flanking region plus 1000 internal and homologous or complementary SEQ ID N °: 21 and SEQ ID N °: 22 constitute an embodiment of this invention. The DNA sequences comprising the new transgene / flanking insertion region, SEQ ID NO: 21, constitute an embodiment of this invention. DNA sequences comprising a sufficient length of polynucleotides of the transgene insert sequence and a sufficient length of polynucleotides of the genomic and / or corn flanking sequence of the DAS maize plant are included. 59122-7 of SEQ ID NO: 21 which are useful as primer sequences for the production of a diagnostic amplicon of the maize plant DAS-59122-7. In addition, DNA sequences comprising the new transgene / flanking insertion region, SEQ ID NO: 22 are provided. DNA sequences comprising a sufficient length of polynucleotides of the transgene insert sequence and a sufficient length are also included. of polynucleotides of the genomic and / or corn flanking sequence of the corn plant DAS-59122-7 of SEQ ID NO: 22 which are useful as primer sequences for the production of a diagnostic amplicon for the corn plant DAS-59122-7. According to another embodiment of the invention, the DNA sequences comprising at least 11 or more nucleotides of the transgene portion of the DNA sequence of SEQ ID NO: 21, or complements thereof, and a similar length of the maize 5 'flanking DNA sequence of SEQ ID NO: 21, or complements thereof, are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for the corn event DAS-59122-7. Therefore, embodiments of the invention also include amplicons produced with homologous or complementary DNA primers of SEQ ID NO: 21.

According to another embodiment of the invention, the DNA sequences comprising at least 11 or more nucleotides of the transgene portion of the DNA sequence of SEQ ID NO: 22, or complements thereof, and a similar length of the 3 'flanking DNA sequence of maize of SEQ ID NO: 22 or complements thereof, are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for the corn event DAS-59122-7. Therefore, embodiments of the invention also include amplicons produced with DNA primers homologous or complementary to SEQ ID NO: 22. More specifically, a pair of DNA molecules comprising a set of DNA primers, wherein the molecules of DNA are identified as SEQ ID N °: 18 or complements thereof and SEQ ID N °: the complements thereof; SEQ ID N °: 2 or complements thereof and SEQ ID N °: 17 or complements thereof; SEQ ID N °: 10 or complements thereof and SEQ ID N °: 9 or complements thereof; SEQ ID N °: 8 or complements thereof and SEQ ID N °: 17 or complements thereof; and SEQ ID N °: 36 or complements thereof and SEQ ID N °: 37 or complements thereof, constitute embodiments of the invention. Other aspects of the invention include an amplicon comprising the DNA molecules of SEQ ID NOS: 18 and SEQ ID N °: 1; the amplicon comprising the DNA molecules of SEQ ID NO: 2 and SEQ ID NO: 17; the amplicon comprising the DNA molecules of SEQ ID NOS: 10 and SEQ ID NOS: 9; and the amplicon comprising the DNA molecules of SEQ ID NOS: 8 and SEQ ID NOS: 17; and the amplicon comprising the DNA molecules of SEQ ID No.:36 and SEQ ID No.:37. Other embodiments of the invention include the following primers, which are useful for detecting or characterizing the event DAS-59122-7 : SEQ ID N °: 11 or complements thereof; SEQ ID N °: 5 or complements thereof; SEQ ID N °: 4 or complements thereof; SEQ ID N °: 7 or complements thereof; SEQ ID N °: 6 or complements thereof; SEQ ID N °: 3 or complements thereof; SEQ ID N °: 18 or complements thereof; SEQ ID N °: 14 or complements thereof; SEQ ID N °: 13 or complements thereof; SEQ ID N °: 15 or complements thereof; SEQ ID N °: 17 or complements thereof; SEQ ID N °: 16 or complements thereof; and SEQ ID N °: 12 or complements thereof. Other embodiments also include the amplicons produced by apparition of any of the primers listed above. According to another embodiment of the invention, methods are provided to detect the presence of a DNA molecule corresponding to the event DAS-59122-7 in a sample, where said methods comprise: (a) putting the sample in comprises DNA extracted from a maize plant in contact with a DNA probe, where the molecule hybridizes under conditions of severe hybridization with the DNA extracted from the corn event DAS-59122-7 and does not hybridize under severe hybridization conditions with the DNA of a control corn plant; (b) subjecting the sample and the probe to severe hybridization conditions; and (c) detect hybridization of the probe with the DNA. More specifically, a method is provided for detecting the presence of a DNA molecule corresponding to event DAS-59122-7 in a sample, wherein said method comprises: (a) putting the sample comprising DNA extracted from a maize plant in contact with a probe of a DNA molecule consisting of sequences that are unique to the event, for example binding sequences, where said DNA probe molecule is hybridized under severe hybridization conditions to the DNA extracted from the DAS maize event -59122-7 and does not hybridize under severe hybridization conditions with the DNA of a control maize plant; (b) subjecting the sample and the probe to severe hybridization conditions; and (c) detect hybridization of the probe with the DNA. In addition, a set of elements and methods is provided to identify the event DAS-59122-7 in a biological sample that detects a specific region of DAS-59122-7 within SEQ ID N °: 23.

DNA molecules are provided which comprise at least one DAS-59122-7 binding sequence selected from the group consisting of SEQ ID N °: 32, 33, 34 and 35 and complements thereof; wherein said binding sequence encompasses the binding between the heterologous DNA inserted in the genome and the DNA that flanks the insertion site in the maize cell, i.e. the flanking DNA and is diagnostic for event DAS-59122-7. According to another embodiment of the invention, methods are provided for producing an insect resistant maize plant comprising the steps of: (a) sexually crossing a first maize progenitor line comprising the expression cassettes of the invention, which confer resistance to insects and a second maize progenitor line that lacks resistance to insects, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that is insect resistant. Such methods may optionally comprise the additional step of backcrossing the progeny plant with the second maize progenitor line to produce pure culture maize plants that are insect resistant. A further embodiment of the invention provides a method for producing an insect resistant maize plant comprising transforming a maize cell with the DNA construct PHI17662A (SEQ ID NO: 24), cultivating the Corn cell transformed into a corn plant, select the corn plant that shows resistance to insects and grow that corn plant to obtain a fertile maize plant. The fertile maize plant can self-pollinate or cross with compatible maize varieties to produce insect-resistant progeny. Another embodiment of the invention is further related to a set of elements for detecting DNA to identify the event of maize DAS-59122-7 in biological samples. The set of elements of the invention comprises a first primer that specifically recognizes the 5 'or 3' flanking region of DAS-59122-7 and a second primer that specifically recognizes a sequence within the foreign DNA of DAS-59122-7 or within the Flanking DNA, for use in a PCR identification protocol. Another embodiment of the invention relates to a set of elements for identifying the event DAS-59122-7 in biological samples, where said set of elements comprises a specific probe having a sequence corresponding to, or complementary to, a sequence presenting between 80% and 100% sequence identity with a specific region of event DAS-59122-7. The sequence of the probe corresponds to a specific region comprising part of the 5 'or 3' flanking region of event DAS-59122-7. The methods and sets of elements that the Embodiments of the present invention may be used for different purposes such as, for example, the following: to identify the event DAS-59122-7 in plants, a plant material or in products such as, for example, feed or food products ( fresh or processed) that comprise the plant material or that derive from it; In addition, or alternatively, the methods and sets of elements can be used to identify a transgenic plant material in order to differentiate a transgenic material from a non-transgenic material; In addition, or alternatively, the methods and sets of elements can be used to determine the quality of the plant material comprising the corn event DAS-59122-7. The sets of elements may also contain the reagents and materials necessary to carry out said detection method. Another embodiment of this invention relates to the corn plant DAS-59122-7 or parts thereof, including, for example, pollen, ovules, vegetative cells, the nuclei of pollen cells and the nuclei of egg cells of the corn plant DAS-59122-7 and the progeny derived from it. The corn seed and plant DAS-59122-7 from which the DNA primer molecules that provide a specific amplicon product are an embodiment of the invention. The preceding aspects and other aspects of the invention will be more apparent from the following detailed description and attached figure. DESCRIPTION OF THE DRAWINGS. FIG. 1. DNA sequence (SEQ ID No.:23) where the transgenic insert PHI17662A is shown, as well as the sequences flanking said transgenic insert. The regions of the 5 'and 3' edges, bp 1 to bp 2593 and bp 9937 to bp 11922 respectively, are underlined. There are two nucleotide differences (bp 6526 and bp 6562) based on a comparison with the transforming plasmid PHP17662 that are highlighted and underlined. FIG. 2. Schematic diagram of the region of the insert in the event DAS-59122-7 Cry34 / 35Abl B.t. divided into three separate sections; the region of the 5 'border with the corn genomic DNA, the intact T-DNA insert and the 3' border region with the corn genomic DNA. The two arrows at the bottom of the insert diagram indicate the start and end points of the sequence derived from the displaced fragments of the 5 'and 3' genome. The other boxes in the lower part of the insert diagram represent PCR fragments that were amplified from genomic DNA of event DAS-59122-7 and sequenced to encompass the intact T-DNA insert and the insert / border binding regions. 5 'and 3'. FIG. 3. Schematic diagram of the region of inserted in the event DAS-59122-7 Cry34 / 35Abl B.t. divided into three separate sections; the 5 'border region with the corn genomic DNA, the intact T-DNA insert and the 3' border region with the maize genomic DNA. The boxes at the bottom of the insert diagram represent PCR fragments located either in the genomic regions of the margins or through the 5 'and 3' junction regions of the T-DNA insert with the maize genomic DNA that were amplified from the genomic DNA of event DAS-59122-7. DETAILED DESCRIPTION OF THE INVENTION. The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in the practice of the present invention. Unless otherwise indicated, the terms should be interpreted according to the conventional use given by the relevant technical specialists. Definitions of common terms of molecular biology can also be found in Rieger et al., Glossary de Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases indicated under 37 CFR § 1822 is used. As used herein, the term "comprises" means that "includes but It is not limited to ".

As used herein, the term "corn" means Zea mays and includes all plant varieties that can be grown with corn, including wild corn species. As used herein, the term "specific to DAS-59122-7" refers to a nucleotide sequence that is suitable to discriminately identify event DAS-59122-7 in plants, plant material or in products such as, for example, feed or food products ( fresh or processed) that comprise, or derive from, plant material. As used herein, the terms "insect resistant" and "affecting insect pests" refer to making changes in the feeding, growth and / or behavior of insects at any stage of development, including for example: killing the insect; delay growth; impede the reproductive capacity; inhibit feeding; and similar. As used herein, the terms "pesticidal activity" and "insecticidal activity" are used as synonyms to refer to the activity of an organism or a substance (such as, for example, a protein) that can be measured using various parameters including, for example, mortality of the pest, loss of weight of the pest, attraction of the pest, repellency of the pest and other physical and behavioral changes of a pest after feeding and / or being exposed to the organism or substance for an appropriate time. For example, "pesticidal proteins" are proteins that exhibit pesticidal activity by themselves or in combination with other proteins. A "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. As used herein, the terms "encoding" or "encoded by", when used in the context of a specific nucleic acid, means that the nucleic acid comprises the information necessary to guide the translation of the sequence of the nucleic acid. nucleotides until obtaining a specific protein. The information that encodes the protein is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within the translated regions of the nucleic acid or may lack such intervening untranslated sequences (e.g., as in the cDNA). A "gene" refers to a fragment of nucleic acid that expresses a specific protein, including the regulatory sequences that precede (5 'non-coding sequences) and that follow (3' non-coding sequences) the coding sequence. A "native gene" refers to a gene as it is found in nature with its own regulatory sequences. A "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found in nature. Accordingly, the chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources or regulatory sequences and coding sequences that are derived from the same source, but arranged in a different manner than in nature. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. The term "strange" refers to a material that is not normally found in the location of interest. Accordingly, the term "foreign DNA" can comprise both recombinant DNA as well as newly rearranged DNA from the plant. A "foreign" gene refers to a gene that is not normally found in the host organism, but that is introduced into that host organism by gene transformation. Foreign genes can comprise native genes inserted into a non-native organism or they can comprise chimeric genes. A "transgene" is any gene that is introduced into the genome of an organism by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted can be termed "insertion site" or "site of interest or target".

As used herein, "insert DNA" refers to the heterologous DNA within the expression cassettes that is used to transform the plant material, as the "flanking DNA" may be the genomic DNA naturally present in an organism. , such as a plant, or foreign DNA (heterologous) introduced by a transformation process that is foreign to the DNA molecule of the original insert, eg, fragments associated with the transformation event. A "flanking region" or "flanking sequence", as used herein, refers to a sequence of at least twenty (20) base pairs, preferably at least fifty (50) base pairs and up to five thousand ( 5000) base pairs which is located either immediately 5 'of and contiguous to or immediately 3' of and contiguous to the DNA molecule of the original foreign insert. Transformation procedures that lead to a random integration of the foreign DNA will result in transformants containing different characteristic and unique flanking regions for each transformant. When recombinant DNA is introduced into a plant by means of a traditional cross, the flanking regions thereof generally do not change. The transformants will also contain unique junctions between an extension of the heterologous DNA of the insert and the genomic DNA or two (2) extensions of genomic DNA or two (2) extensions of heterologous DNA. A "union" is the point where two (2) specific DNA fragments join. For example, there is a junction where the insert DNA binds to the flanking DNA. There is also a point of attachment in a transformed organism where two (2) DNA fragments are joined together in a manner that is modified with respect to the form found in the native organism. "Binding DNA" refers to DNA that comprises a point of attachment. As used herein, the term "heterologous" with reference to a nucleic acid is a nucleic acid originating from a foreign species or if from the same species, is substantially modified with respect to its native form as to composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous nucleotide sequence may be of a species different from the species from which it derived the nucleotide sequence or if it is from the same species, the promoter is not operably linked naturally to the sequence of nucleotides. The heterologous protein may be native to a foreign species or if it is of the same species, it is substantially modified with respect to its original form by deliberate human intervention. The "regulatory sequences" refer to nucleotide sequences located 5 '(sequences not 'encoders), within or 3' (3 'non-coding sequences) of a coding sequence and affecting transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences may include promoters, translational leader sequences, introns and polyadenylation recognition sequences. A "promoter" refers to a nucleotide sequence that has the ability to control the expression of a functional RNA or coding sequence. In general, the coding sequence is located 3 'with respect to the promoter sequence. The sequence of the promoter consists of elements towards the proximal and more distal 5 'end, the latter elements being called enhancers. Accordingly, an "enhancer" is a nucleotide sequence that can stimulate the activity of the promoter and can be an innate element of the promoter or a heterologous element inserted to increase the level or specificity of tissues of a promoter. The promoters can derive in their entirety from a native gene or they can be composed of different elements derived from different promoters present in nature or they can even comprise segments of synthetic nucleotides. Those skilled in the art will understand that different promoters can direct the expression of a gene in different tissues or cell types, at different stages of development or as a response to different environmental conditions. Promoters that allow a nucleic acid fragment to be expressed in most cell types at any time are commonly referred to as "constitutive promoters". New promoters of various types that are useful in plant cells are continuously being discovered; numerous examples can be found in the compilation of Okamuro and Goldberg (1989) Biochemistry of Plants 15: 1-82. It is further considered that since in most cases the exact boundaries of the regulatory sequences have not been completely defined, fragments of nucleic acids of different lengths may exhibit identical promoter activity. The "leader sequence of the translation" refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The leader sequence of the translation is present in the fully processed mRNA towards the 5 'end of the translation start sequence. The leader sequence of the translation may affect one or more of the following: processing of the primary transcript in mRNA, stability of the mRNA and translation efficiency. Examples of direct translation sequences have been described (Turner and Foster (1995) Mol Biotechnol 3: 225-236).

"Non-coding sequences 3 '" refer to nucleotide sequences located towards the 3' end of the coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression . The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid extensions to the 3 'end of the mRNA precursor. The use of different 3 'non-coding sequences can be found, for example, in Ingelbrecht et al. (1989) Plant Cell 1: 671-680. A "protein" or "polypeptide" is a chain of amino acids arranged in a specific order determined by the coding sequence of the polynucleotide encoding said polypeptide. A DNA construct is an assembly of DNA molecules linked together that provide one or more expression cassettes. The DNA construct can be a plasmid capable of self-replicating in a bacterial cell and contains numerous sites of restriction endonuclease enzymes that are useful for introducing DNA molecules that provide functional genetic elements, ie, promoters, introns, directives, sequences of coding, 3 'termination regions, among others; or the DNA construction can be a linear assembly of DNA molecules, such as an expression cassette. The expression cassette contained in the DNA construct comprises the genetic elements necessary to allow the transcription of a messenger RNA. The expression cassette can be designed to be expressed in prokaryotic cells or eukaryotic cells. The expression cassettes of the embodiments of the present invention are designed to be expressed in plant cells. The DNA molecules of the embodiments of the invention are provided in expression cassettes for expression in the organism of interest. The cassette will include 5 'and 3' regulatory sequences operatively linked to the coding sequence. The term "operably linked" means that the ligated nucleic acid sequences are contiguous and when it is necessary to bind the coding regions of two proteins, they are contiguous and in the same reading frame. The term "operably linked" refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and intervenes in the transcription of the DNA sequence corresponding to said second sequence. The cassette may also contain at least one additional gene to be cotransformed in the organism. Alternatively, one or more additional genes may be provided in multiple expression cassettes or multiple DNA constructs.

The expression cassette will include in the transcription direction 5 'to 3': a transcription and translation initiation region, a coding region and a transcription termination region and functional translation in the host organism. The region of initiation of transcription (ie, the promoter) may be native or analogous, or foreign or heterologous to the host organism. In addition, the promoter can be the natural sequence or, alternatively, a synthetic sequence. The expression cassettes may also contain 5 'direct sequences in the construction of the expression cassette. Said guidelines sequences can act to increase translation. It should be noted that, as used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of a heterologous nucleic acid including transgenic altered initially, as well as those created by sexual crossings or asexual propagation from the initial transgenic. The term "transgenic", as used herein, does not cover genome alteration (chromosomal or extrachromosomal) by conventional plant breeding methods or by natural events, such as cross-fertilization, non-recombinant viral infection, transformation bacterial non-recombinant, transposition not recombinant or spontaneous mutation. A transgenic "event" is produced by transformation of plant cells with one or more heterologous DNA constructs, including an expression cassette with a nucleic acid comprising a transgene of interest, the regeneration of the plant population that is the result of the insertion of the transgene in the genome of the plant and selection of a particular plant characterized by the insertion of it in a particular location of the genome. An event is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic arrangement of a plant. The term "event" also refers to the progeny produced by a sexual cross between the transformant and another variety that includes the heterologous DNA. Even after a repeated backcrossing with a recurrent parent, the inserted DNA and the flanking DNA of the transformed parent are present in the progeny of the crossing at the same chromosomal location. The term "event" also refers to the DNA of the original transformant comprising the inserted DNA and the immediately adjacent flanking sequence of the inserted DNA that is expected to be transferred to the progeny that will receive the inserted DNA, including the transgene of interest as a result of sexual crossing of a progenitor line that includes the inserted DNA (for example, the transformant original and progeny resulting from self-crossing) with a parent line that does not contain the inserted DNA. The insect-resistant maize plant DAS-59122-7 can be bred with a first sexual crossing of a first progenitor maize plant consisting of a maize plant grown from the transgenic DAS-59122-7 maize plant and the progeny thereof, derived from the transformation with the expression cassettes of the embodiments of the present invention that confer resistance to insects and a second progenitor maize plant without insect resistance thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is insect resistant; and autocrossing the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from among the second progeny plants an insect resistant plant. These steps may also include the backcrossing of the first insect-resistant progeny plant or the second insect-resistant progeny plant with the second parent corn plant or a third parent corn plant, thereby producing a corn plant that It is resistant to insects. As used herein, the term "plant" refers to whole plants, plant organs (by example, leaves, stems, roots, etc.), seeds, plant cells and progeny thereof. The parts of transgenic plants that are considered to be within the scope of the invention comprise, for example, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots originating from the transgenic plants or the progeny of the same ones previously transformed with a DNA molecule of the invention and which therefore consist, at least in part, of transgenic cells, also constitute an embodiment of the present invention. As used herein, the term "plant cell" includes, for example, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants that is amenable to transformation techniques, including monocotyledonous and dicotyledonous plants. The term "transformation" refers to the transfer of a nucleic acid fragment to a genome of a host organism, resulting in a genetically stable inheritance. The host organisms that contain the transformed nucleic acid fragments are referred to as "transgenic organisms". The examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol 143: 277) and accelerated particle transformation technology or "gene gun" (Klein et al., 1987) Nature (London) 327: 70-73; U.S. Patent No.: 4,945,050, incorporated herein by reference). Further transformation methods are described below. Accordingly, the isolated polynucleotides of the invention can be incorporated into recombinant constructs, typically DNA constructs, capable of being introduced and replicated in a host cell. Said construction may be a vector that includes a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide into a given host cell. Numerous vectors suitable for stable transfection of plant cells or for establishing transgenic plants have been described, for example, Pouwels et al., (1985; Sup. 1987) Cloning vectors: A Laboratory Manual, Weissbach and eissbach (1989) Methods for Plant Molecular Biology, (Academic Press, New York); and Flevin et al., (1990) Plant Molecular Biology Manual, (Kluwer Academic Publishers). Typically, plant expression vectors include, for example, one or more cloned plant genes under the control of transcription of 5 'and 3' regulatory sequences. and a dominant selectable marker. Said plant expression vectors may also contain a promoter regulatory region (eg, a regulatory region that controls an inducible or constitutive expression, regulated by the environment or by the development or specificity of cells or tissues), a transcription initiation site , a ribosome binding site, an RNA processing signal, a transcription termination site and / or a polyadenylation signal. It should also be taken into account that it is also possible to cross two different transgenic plants to produce an offspring that contains two exogenous genes, aggregates of independent segregation. Autocrossing of the appropriate progeny can produce plants that are homozygous for both introduced exogenous genes. Backcrossing with a parent plant and crossing with a non-transgenic plant, as well as vegetative propagation, is also contemplated. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in any of the numerous references, for example, Fehr, in ethods of Breeding for Cultivation Development, Wilcos J. ed., American Society of Agronomy , Madison is. (1987). A "probe" is an isolated nucleic acid to which a conventional detectable label or molecule has been attached informant, for example, a radioactive isotope, a ligand, a chemiluminescent agent or an enzyme. Said probe is complementary to a chain of the nucleic acid of interest, in the case of the present invention, of a genomic DNA chain of the corn event DAS-59122-7, either from a maize plant or from a sample that includes the DNA of the event. The probes according to the present invention not only include deoxyribonucleic or ribonucleic acid but also polyamides and other probe materials that specifically bind to the DNA sequence of interest and that can be used to detect the presence of said DNA sequence of interest. The "primers" are isolated nucleic acids that are cold aligned with a complementary DNA strand of interest by hybridizing the nucleic acid to form a hybrid between the primer and the DNA strand of interest, then extending along the length of the strand. the DNA chain of interest by action of a polymerase for example, a DNA polymerase. The primer pairs of the invention are intended for the amplification of a target nucleic acid sequence for example, by polymerase chain reaction (PCR) or other conventional methods of nucleic acid amplification. "PCR" or "polymerase chain reaction" is a technique used for the amplification of specific segments of DNA (see, US Patents Nos .: 4,683,195 and 4,800,159; incorporated in this document as a reference). The probes and primers are of sufficient nucleotide length to bind to the DNA sequence of interest specifically under the conditions of hybridization or the reaction conditions determined by the operator. This length can be any length that is sufficient to be useful in the detection method of choice. In general, eleven (11) nucleotides or more in length, preferably eighteen (18) nucleotides or more and more preferably twenty-two (22) nucleotides or more are used. Said probes and primers are specifically hybridized with a white sequence under conditions of hybridization of great severity. The probes and primers according to embodiments of the present invention can have a complete DNA sequence similarity to the contiguous nucleotides of the sequence of interest, although it is possible to design probes that differ from the DNA sequence of interest and that retain the ability to hybridize with the DNA sequence of interest by conventional methods. Probes can be used as primers, but are generally designed to bind to the DNA or RNA of interest and not for an amplification process. Specific primers can be used to amplify an integration fragment in order to produce an amplicon that can be used as a "specific probe" to identify the DAS-59122-7 event in biological samples. When the probe hybridizes with the nucleic acids of a biological sample under conditions that allow the binding of the probe with the sample, this binding can be detected and therefore may be indicative of the presence of the event DAS-59122-7 in the sample biological The identification of the probe junction is described in the art. In one embodiment of the invention, the specific probe is a sequence that, under optimal conditions, hybridizes specifically to a region within the 5 'or 3' flanking region of the event and also comprises a portion of the foreign DNA contiguous therewith. The specific probe can comprise a sequence of at least 80%, between 80 and 85%, between 85 and 90%, between 90 and 95% and between 95 and 100% identical (or complementary) to a specific region of the event. Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. , vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 (hereinafter, "Sambrook et al., 1989"); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, "Ausubel et al., 1992"); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. Primer pairs for PCR can be derived from a known sequence, for example, using computer programs intended for that purpose, such as the PCR Primer Analysis Tool program in Vector NTI version 6 (Informax Inc., Bethesda MD); PrimerSelect (DNASTAR Inc., Madison, WI); and Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). In addition, the sequence can be scanned visually and the primers can be manually identified using the guidelines known to those skilled in the art. A "set of elements", as used herein, refers to a set of reagents intended to carry out embodiments of methods of the invention, more particularly, the identification of event DAS-59122-7 in biological samples. . The set of elements of the invention can be used, and the components can be specifically adjusted, for a quality control (for example, the purity of seed lots), the detection of event DAS-59122-7 in a plant material or in a material comprising or deriving from plant material, such as, for example, feed or food products. A "plant material", as used herein, refers to a material that is obtained or derived from a plant.

The primers and probes based on the flanking DNA and the inserted sequences described herein can be used to confirm (and, if necessary, to correct) the described sequences by conventional methods, for example, by recloning and sequencing said sequences. The probes and nucleic acid primers of the present invention are hybridized under stringent conditions to the DNA sequence of interest. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample. The nucleic acid molecules or fragments thereof have the ability to hybridize specifically with other nucleic acid molecules under certain circumstances. As used herein, it is said that two nucleic acid molecules have the ability to specifically hybridize with each other, if the two molecules can form a double-stranded, antiparallel nucleic acid structure. It is said that a nucleic acid molecule is "Complementary" of another nucleic acid molecule if they have complete complementarity. As used herein, molecules are considered to have "complete complementarity" when each nucleotide of one of the molecules is complementary to one nucleotide of the other.

It is said that two molecules are "minimally complementary" if they can be hybridized with each other "with sufficient stability to remain cold aligned with each other under at least conventional" low severity "conditions. Similarly, it is considered that the molecules are "complementary" if they hybridize to each other with sufficient stability to remain cold aligned with each other under conventional "high severity" conditions. Conventional severity conditions were described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985); therefore, deviations from complete complementarity are possible, provided that such deviations do not completely impede the ability of the molecules to form a double-stranded structure. For a nucleic acid molecule to serve as a primer or probe, it is only necessary that it be sufficiently sequentially complementary to form a stable double-stranded structure under the particular concentrations of solvent and salts employed. In hybridization reactions, the speci fi city is typically a function of the post-hybridization washes, the critical factors being the ionic strength and the temperature of the final wash solution. The thermal melting point (Tm) is the temperature (under an ionic strength and defined pH) at which 50% of a complementary sequence of interest is hybridized with a perfectly matching probe. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1984), Anal. Biochem. , 138: 267-284: Tm = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500/1; where M is the molarity of the monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. The Tm is reduced by approximately 1 ° C for every 1% of mismatch; therefore, it is possible to adjust the Tm, the hybridization and / or washing conditions to hybridize the sequences of the desired identity. For example, if you search for sequences with > 90% identity, the Tm can be decreased by 10 ° C. In general, severe conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, very severe conditions may employ hybridization and / or washing at 1, 2, 3 or 4 ° C less than Tm; moderately severe conditions may utilize hybridization and / or wash at 6, 7, 8, 9 or 10 ° C less than Tm; the conditions of low severity can employ a hybridization and / or a wash at 11, 12, 13, 14, 15 or 20 ° C less that the Tm. Using the equation, the hybridization and washing compositions and the desired Tm, the skilled artisan will understand that variations in the severity of the hybridization and / or wash solutions are inherently described. If the degree of mismatch desired results in a Tm less than 45 ° C (aqueous solution) or 32 ° C (formamide solution) it is preferred to increase the concentration of SSC so that a higher temperature can be used. A very extensive guide for the hybridization of nucleic acids can be found in Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acids Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and iley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). As used herein, a substantially homologous sequence is a nucleic acid molecule that will hybridize specifically to the complement of the nucleic acid molecule with which it is being compared under conditions of great severity. Appropriate severity conditions that promote DNA hybridization, for example, sodium chloride / 6X sodium citrate (SSC) at 45 ° C, followed by a wash of 2X SSC at 50 ° C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). 6.3.1-6.3.6. Typically, severe conditions will be those in which the concentration of salts is less than about 1.5 M of the Na ion, typically between about 0.01 and 1.0 M Na (or other salts) ion concentration. pH between 7.0 and 8.3 and the temperature is at least about 30 ° C for short probes (for example, between 10 and 50 nucleotides) and at least about 60 ° C for long probes (for example, more than 50 nucleotides). Severe conditions can also be achieved with the addition of destabilizing agents, such as formamide. Examples of low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C and a 2X SSC IX wash (20X SSC = NaCl 3.0 M / 0.3 M trisodium citrate) at 50-55 ° C. Examples of moderate severity conditions include hybridization in formamide 40 to 45%, 1 M NaCl, 1% SDS at 37 ° C and a 0.5X to IX SSC wash at 55-60 ° C. Examples of high severity conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C and a wash in 0.1X SSC at 60-65 ° C. A nucleic acid of the invention can be specifically hybridized with one or more of the unique nucleic acid molecules for the event DAS-59122-7, or complements thereof or fragments of any of them under moderately severe conditions. Methods of sequence alignment for comparison are well known in the art. Accordingly, the determination of the percentage identity between any two sequences can be performed using a mathematical algorithm. Preferred and non-limiting examples of said mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math, 2: 482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol Biol. 48: 443-453; the similarity search method of Pearson and Lipman (1988) Proc. Nati Acad. Sci. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. Sci. USA 87: 2264, modified as described in Karlin and Altschul (1993) Proc. Nati Acad. Sci. USA 90: 5873-5877. The computerized implementations of these mathematical algorithms can be used for the comparison of sequences in order to determine the sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0); the ALIGN PLUS program (version 3.0, copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 10 (available from Accelrys, 9685 Scranton Road, San Diego, CA 92121, USA). Alignments using these programs can be made using the default parameters. The CLUSTAL program is well described in Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The ALIGN and ALIGN PLUS programs are based on the algorithm of Myers and Miller (1988) supra. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. The BLAST family of programs that can be used for database similarity searches includes: BLASTN to query nucleotide sequences in nucleotide sequence databases; BLASTX for the query of nucleotide sequences in databases of protein sequences; BLASTP for the query of protein sequences in protein sequence databases; TBLASTN for the query of protein sequences in the database of nucleotide sequences; and TBLASTX for queries of nucleotide sequences in databases of nucleotide sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds. , Greene Publishing and Wiley-Interscience, New York (1995). The alignment can also be done manually by visual inspection. In order to obtain alignments with mismatches to make a comparison, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that allows detecting distant relationships between the molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST, PSI-BLAST, the predetermined parameters of the respective programs can be used (eg.), BLASTN for nucleotide sequences, BLASTX for proteins). See www. ncbi. hlm. nih go As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences that are the same when aligned by maximum correspondence in the specified comparison window. When the percentage of sequence identity is used with reference to proteins it will be understood that the positions of the residues that are not identical often differ by conservative amino acid substitutions, where the amino acid residues are substituted by other amino acid residues with similar chemical properties (eg, charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When the sequences differ by conservative substitutions, the percentage of sequence identity can be adjusted upward in order to correct the conservative nature of the substitution. It is said that the sequences that differ by said conservative substitutions possess "sequence similarity" or "similarity". The means for effecting this adjustment are well known to those skilled in the art. Typically, it involves qualifying a conservative substitution as a partial rather than a complete mismatch, thereby increasing the percentage of sequence identity. Thus, for example, when an identical amino acid receives a score of 1 and a non-conservative substitution receives a score of zero, the conservative substitution receives a score between zero and 1. The rating of conservative substitutions is calculated, for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, California). As used herein, the "percent sequence identity" means the value determined by comparison of two optimally aligned sequences in a comparison window, where the portion of a sequence of polynucleotides in the comparison window may comprise additions or deletions (i.e., mismatches) compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result per 100 to obtain the percentage of sequence identity.- With respect to the amplification of the target nucleic acid sequence (for example by PCR) with a pair of determined amplification primers, the "severe conditions" are those conditions that allow the pair of primers to only hybridize to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or complement thereof) would be attached to preferably produce a single amplification product, the amplicon , in a thermal amplification reaction of DNA. The term "specific for (a sequence of interest or blank)" indicates that the probe or primer hybridizes under stringent hybridization conditions with the target sequence only in a sample comprising said target sequence. As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid annealing. For example, to determine if the maize plant that is the result of a sexual cross contains the transgenic genomic DNA transgenic event of a maize plant of the invention, the extracted DNA can be subjected to a tissue sample from the plant of maize to a nucleic acid amplification method using a pair of DNA primers including a first primer derived from the flanking sequence in the plant genome adjacent to the insertion site of the heterologous inserted DNA and a second primer derived from the heterologous inserted DNA to produce an amplicon that is diagnostic of the presence of the DNA event. Alternatively, the second primer can be derived from the flanking sequence. The amplicon is of a length and has a sequence that is also diagnostic of the event. The amplicon can vary in length from the combined length of the primer pairs plus a pair of nucleotide bases to any length of amplicon that can be produced with a DNA amplification protocol. Alternatively, the pair of primers can be derived from the flanking sequences on both sides of the inserted DNA to produce an amplicon including the entire nucleotide sequence of the expression construct PHI17662A as well as the sequence flanking the transgenic insert, see FIG. 1 (SEQ ID NO: 23), of a size of approximately 12 (twelve) Kb. One of the members of the pair of primers derived from the genomic sequence of the plant can be located at a distance from the inserted DNA sequence, and this distance can vary in a range of one nucleotide up to the limits of the amplification reaction or about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that can be formed in the thermal amplification reaction of DNA. The amplification of the nucleic acid can be carried out by any of the various nucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). A wide variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos .: 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods were developed to amplify up to 22 kb of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Nati Acad. Sci. USA 91: 5695-5699, 1994). These methods, as well as other methods known in the art of DNA amplification, can be employed in the practice of the embodiments of the present invention. It is considered that it may be necessary to adjust numerous parameters in a specific PCR protocol in order to meet specific laboratory conditions and can be modified slightly and still obtain similar results. These adjustments will be evident to the art specialist. The amplicon produced with these methods can be detected by a plurality of techniques including, for example, the Genetic Bit Analysis (Nikiforov, et al., Nucleic Acid Res. 22: 4167-4175, 1994) by which an oligonucleotide is designed of DNA that overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in the cavities of a microcavity plate. After PCR of the region of interest (using a primer for the inserted sequence and one for the adjacent flanking genomic sequence), a single chain PCR product can be hybridized with the immobilized oligonucleotide and can serve as a quench for a reaction of a single base extension using a specific labeled DNA polymerase and ddNTP for the next expected base.

The reading can be based on fluorescence or an ELISA. A signal indicates the presence of the insert / flanking sequence due to successful single base amplification, hybridization and extension. Another method is the pyrosequencing technique described by inge (Innov, Phrma, Tech. 00: 18-24, 2000). In this method, an oligonucleotide is designed that overlaps the junction of the adjacent genomic DNA and the binding DNA of the insert. The oligonucleotide is hybridized with a single chain PCR product of the region of interest (one primer for the inserted sequence and one for the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5 'phosphosulfate and luciferin. The DNTPs are added individually and the incorporation results in a light signal that is measured. The light signal indicates the presence of the sequence of the transgenic / flanking insert due to amplification, hybridization and extension of single or multiple bases. The Fluorescence Polarization described by Chen, et al. (Genome Res. 9: 492-498, 1999) is also a method that can be used to detect the amplicon of the invention. The use of this method allows to design an oligonucleotide that overlaps the flanking and inserted genomic DNA binding. The oligonucleotide is hybridized with a simple chain PCR product of the region of interest (one primer for the inserted DNA sequence and one for the flanking genomic DNA) and incubated in the presence of a DNA polymerase and a fluorescently labeled ddNTP. The extension of a single base results in the incorporation of the ddNTP. Said incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgenic / flanking insert sequence due to successful single base amplification, hybridization and extension. Taqman® (PE Applied Biosystems, Foster City, CA) is described as a method for detecting and quantifying the presence of a DNA sequence and is very well explained in the instructions provided by the supplier. Briefly, a FRET oligonucleotide probe is designed that overlaps the junction between the flanking genomic DNA and the insert. The FRET probe and the PCR primers (one primer for the inserted DNA sequence and one for the flanking genomic sequence) are cyclized in the presence of a thermostable DNA polymerase and the dNTPs. Hybridization of the FRET probe results in the hydrolysis or "cleavage" and the release of the fluorescent group from the neutralizing group on the FRET probe. A fluorescent signal is indicative of the presence of the flanking / transgenic insert sequence due to successful amplification and hybridization.

The use of Molecular Beacons [Molecular Markers] for the detection of sequences in Tyangi, et al. (Nature Biotech, 14: 303-109, 1996). Briefly, a FRET oligonucleotide probe is designed that overlaps the junction of the flanking genomic DNA and the insert. The unique structure of the FRET probe allows it to contain a secondary structure that preserves the fluorescent and neutralizing groups next to each other. The FRET probe and primers for PCR (one primer for the DNA sequence of the insert and the other for the genomic flanking sequence) are cycled in the presence of a thermostable polymerase and the dNTPs. After successful PCR amplification, hybridization of the FRET probe with the target sequence results in the elimination of the secondary structure of the probe and the spatial separation of the fluorescent and neutralizing groups. A fluorescent signal is obtained. The fluorescent signal is indicative of the presence of the flanking / transgenic insert sequence due to successful amplification and hybridization. A hybridization reaction with a probe specific for the sequence contained in the amplicon is another method for detecting the amplicon produced by the PCR reaction. Embodiments of the present invention are further defined in the following examples. You must have in note that these examples are only offered for illustrative purposes. From the above description and these examples, the person skilled in the art can understand the essential characteristics of this invention and without departing from the spirit and scope thereof, can make different changes and modifications to the embodiments of the invention in order to adapt it to different uses and conditions. Accordingly, the various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to the person skilled in the art from the foregoing description. Said modifications are also included within the scope of the appended claims. The content of each reference cited herein is incorporated herein in its entirety by way of reference. EXAMPLES Example 1. Transformation of maize by transformation with Agrobacterium and regeneration of transgenic plants containing the genes Cry34Abl and Cry35Abl (Cry34 / 35Abl). To transform the embryonic tissue of maize, a DNA molecule of approximately 7.4 Kb was used. PHI17662A (SEQ ID NO: 24), which includes a first transgenic expression cassette comprising a DNA molecule that includes the promoter, a non-translated 5'-exon and the first intron of the ubiquitin gene (Ubi-1) of corn (Christensen et al. (1992) Plant Mol. Biol. 18: 675-689 and Christensen and Quail (1996) Transgenic Res. 5: 213-218) operatively linked to a DNA molecule that codes for a B-d-endotoxin identified as Cry34Abl (U.S. Patent Nos .: 6,127,180, 6,624,145 and 6,340,593) operatively linked to a DNA molecule comprising a Pin II transcription terminator isolated from potato (Gyheung An et al. (1989) Plant Cell 1: 115-122). The second cassette of transgenic expression of the DNA construct comprises a DNA molecule encoding the wheat peroxidase promoter (Hertig et al. (1991) Plant Mol. Biol. 16: 171-174) operably linked to a DNA molecule. which codes for a Bt d-endotoxin identified as Cry35Abl (U.S. Patent Nos .: 6,083,499, 6,548,291 and 6,340,593) operatively linked to a DNA molecule comprising a Pin II transcription terminator isolated from potato (Gyheung An et al. (1989) Plant Cell 1: 115-122). The third cassette of transgenic expression of the DNA construct comprises a DNA molecule of the 35S promoter of the cauliflower mosaic virus (CaMV) (Odell JT et al (1985) Nature 313: 810-812; Mitsuhara et al. 1996) Plant Cell Physiol. 37: 49-59) operably linked to a DNA molecule encoding a phosphinothricin acetyltransferase (PAT) gene (Wohlleben W. et al. (1988) Gene 70: 25-37) operably linked to a molecule of DNA comprising a 3 'transcription terminator of 35S (CaMV) (see Mitsuhara et al (1996) Plant Cell Physiol. 37: 49-59). Abl Cry34 / 35 B.t. maize plants were obtained. by transformation with Agrobacterium, using the Zhao method (U.S. Patent No. 5,981,840, and PCT patent publication W098 / 32326, the contents of which are incorporated herein by reference). Briefly, immature maize embryos were isolated and the embryos were contacted with a suspension of Agrobacterium, where the bacteria were able to transfer the DNA PHI17662 (SEQ ID N0: 24) to at least one cell of at least one of the embryos. immature (step 1: the infection step). Specifically, in this step immature embryos were immersed in a suspension of Agrobacterium to initiate inoculation. The embryos were co-cultivated for a time with the Agrobacterium (step 2: the step of cocultivation). Specifically, the immature embryos were cultured on a solid medium after the infection step. After this period of co-cultivation a step of "rest" was applied. In this resting step, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for the plant transformants (step 3: resting step). In particular, immature embryos are grown on a solid medium with antibiotic, but without a selection agent, to eliminate the Agrobacterium and to provide a resting phase to the infected cells. Next, the inoculated embryos were cultured on a medium containing a selective agent and the transformed calluses in growth were recovered (step 4: the selection step). Specifically, the immature embryos were cultured on a solid medium with a selective agent resulting in the selective growth of the transformed cells. Then, the calluses were regenerated in plants (step 5: the regeneration step) and, specifically, the calluses grown on selective medium were grown on solid medium to regenerate the plants. During culture, the individual embryos were kept physically separated and most of the explants died on the selective medium. Those embryos that survived and produced healthy callus tissue resistant to glufosinate received unique identification codes that represented putative transformation events and were continuously transferred to fresh selection medium. Plants were regenerated from the tissue derived from each single event and then transferred to the greenhouse. Leaf samples were taken for molecular analysis in order to verify the presence of the transgene by PCR and to confirm the expression of the Cry34 / 35Abl protein by ELISA. Next, the plants were subjected to a bioassay with whole plants using western corn rootworms. The positive plants were crossed with inbred lines to obtain seeds of the initial transformed plants. Numerous lines were evaluated in the field. Event DAS-59122-7 was selected from a population of independent transgenic events based on a combination of superior characteristics, including insect resistance and agronomic performance. Example 2. Identification of corn line DAS-59122-7 Cry34 / 35Abl Bacillus thuringiensis Seeds of event DASD59122D7 were evaluated. The seeds T1S2 represent the transformation in the antecedent Hi-II, followed by a crossing with the inbreeding line PH09B and two rounds of self-crossing. All seeds were obtained from Pioneer Hi-Bred (Johnston, IA). The primary characterization was carried out with foliar plant tissue during the study confirming the activity of phosphinothricin acetyltransferase (PAT) by painting the leaves with the herbicide and expressing Cry34Abl using lateral flow devices. In this study the control substances were defined as unmodified seeds representative of the background of the test substance. Seeds were used control with Hi-II and PH09B background as negative controls. These unmodified seeds do not contain the transcription units for the cry34Abl, cry35Abl and pat genes. All seeds were obtained from Pioneer Hi-Bred (Johnston, IA). DNA samples from two events Cry34 / 35Abl B.t. additional, event DAS-45214-4 and event DAS-45216-6, as negative controls for a PCR analysis specific to the event. The two events were produced by transformation with Agrobacterium using the same vector used to produce event DAS-59122-7 and therefore contained the plant transcription units for the genes cry34Abl, cry35Abl and pat. However, the T-DNA insertion sites in events DAS-45214-4 and DAS-45216-6, including the genomic DNA of the border regions, were different from the corresponding in event DAS-59122-7. DNA samples from event DAS-45214-4 and event DAS-45216-6 were isolated and characterized by Southern blot analysis (data not shown.) Corn seeds were seeded for event DAS-59122-7 and unmodified control seeds (Hi-II and PH09B) in growth chambers at the DuPont experimental station (Wilmington, DE) to produce sufficient quantities of plants to analyze the DNA. For the characterization of event DAS-59122-7, ten (10) T1S2 seeds were sown.

Ten (10) seeds were also sown for each unmodified control line. One (1) seed was planted per pot, and the pot was unequivocally identified. The conditions of planting and cultivation were appropriate for the growth of healthy plants, including regulation of light and water. Leaf samples were collected for each of the control plants and event DAS-59122-7. For each sample, sufficient foliar material was collected above the growth point and placed in a pre-labeled sample bag. The samples were placed on dry ice and then transferred to a very low temperature freezer after harvesting. All samples were kept frozen until processing. All the leaf samples were unequivocally labeled with the identifier of the plant and the date of harvest. To confirm the expression of the Cry34Abl protein in the DAS-59122-7 event and the absence of expression in the controls, leaf samples were collected from all the plants of the DAS-59122-7 event and control, and tracked in the same transgenic protein using lateral flow devices specific for Cry34Abl (Strategic Diagnostics, Inc., Newark, DE). Samples of the leaves of each plant were sampled and ground in a phosphate buffered saline solution with Tween 20 to extract the crude protein. A device was dipped with strips in the extract to determine the presence or absence of the Cry34Abl protein. The results of the immunoassay were used to confirm the identity of the test substance in the plants before the molecular analysis as shown in Table 1. To confirm the expression of phosphinothricin acetyltransferase (PAT) in plants of the event DAS-59122-7, the leaves were painted with herbicide. All the plants used in this study were painted on the leaves to confirm the identity of the plant. The plants were evaluated before the growth stage Rl. The tests were conducted using a standard procedure known in the art for the painting of the leaves with herbicide in order to identify transgenic plants expressing PAT. Specifically, a portion of a leaf of each plant was treated with an approximately 2% solution of the herbicide glufosinate, Bastaá (Bayer CropScience) in water and the presence of brown or necrotic tissue was visually checked in the painted area of the leaves. 12 days after the application. The results were recorded for each plant and were used to determine the expression of PAT in each test plant and are shown in Table 1. As shown in Table 1, of the ten (10) plants evaluated by the generation of the event DAS-59122-7 T1S2, six (6) plants expressed both Cry34Abl and PAT, while four (4) plants did not express none of the proteins. All unmodified controls were negative for both CryAbl and PAT assays (data not shown). Table 1: Expression of Cry3 Abl and PAT proteins and Southern hybridization data for event DAS-59122-7 Cry34 / 35Abl B. t.

The positive Cry34Abl expression indicates that there was detection of protein expression determined with the lateral flow device based on immunoassays to detect the Cry34Abl protein. The negative expression indicates that there was no detection of the Cry34Abl protein. The expression positive PAT indicates that the plants were tolerant to the treatment with the herbicide and negative indicates that the plants were sensitive to the herbicide. 2 '+ indicates that there was a hybridization signal with the Southern blot; - indicates that there was no hybridization signal with the Southern blot. The cry34Abl gene probe hybridized with the expected 1915 kb internal T-DNA fragment, the cry35Abl gene probe was hybridized with the expected 2,607 kb internal T-DNA fragment and the pat gene probe was hybridized with a fragment of the 3.4 kb border consistent with a single intact T-DNA insertion determined by Southern blot analysis. Example 3. Southern blot analysis of DAS-59122-7 corn line with Cry34 / 35Abl from Bacill s thuringiensis One gram samples of leaves were milled under liquid nitrogen and genomic DNA was isolated using the DNeasy plant element set ® (Qiagen, Valencia, CA) or using the procedure with standard urea extraction buffer solution. After extraction, the DNA was visualized on an agarose gel to determine the quality thereof and was quantified using spectrofluorometric analysis with the Pico Green® reagent (Molecular Probes, Inc., Eugene, OR). The 1 Kb DNA standard (Invitrogen, Carlsbad, CA) was used to estimate the sizes of the DNA fragments on the agarose gels. The genomic DNA isolated from the plants with the event DAS-59122-7 was digested with Neo I and separated by electrophoresis, transferred to nylon membranes and hybridized with the probes of the cry34Abl, cry35Abl and pat genes using the standard procedures known in art. The transferred ones were exposed to an X-ray film for one or more periods of time to detect the hybridizing fragments and to visualize the molecular weight standards. The images were then digitally captured by taking photographs of the X-ray films and / or by detection with a Lumi-Imager ™ instrument (Roche, Indianapolis, IN). The sizes of the bands detected for each probe were documented. Southern blot analysis was used as a means to verify the presence of the insert in the test plants and confirm that all the plants of the DAS-59122-7 event contained the same insert as shown in Table 1. (not shown data from the Southern transfer.) The Southern transfer analysis indicated that the DAS- event 59122-7 contained a single insert consisting of an intact copy of the T-DNA region of the plasmid PHP17662, while the null segregants determined with the protein expression analysis did not hybridize with the gene probes. In addition, plants with event DAS-59122-7 expressing the two proteins showed identical hybridization patterns with Southern blots (data not shown). Specifically, the cry34Abl gene probe hybridized with the expected 1915 kb internal T-DNA fragment, the cry35Abl gene probe was hybridized with the expected 2,607 kb internal T-DNA fragment and the pat gene was hybridized with a 3.4 kb border fragment consistent with a single intact T-DNA insertion determined according to the Southern blot results. Example 4. Sequencing of the T-DNA insert and of the region flanking the border of Cry34 / 35Abl of Bacillus thuringiensis in the corn line DAS-59122-7 The T-DNA insert and the regions flanking the edges were cloned. of the T-DNA of Cry34 / 35Abl Bt of the event DAS59122-7 using the PCR-based methods diagrammed in Figures 2 and 3. Specifically, the sequences bordering the 5 'and 3' ends of the insert in event DAS-59122-7 were obtained using two displacement techniques per the genome The first method of displacement was essentially the method described for the set of Universal Genome Walker elements (BD Biosciences Clontech, Palo Alto, CA) and the second method was applied according to the splinkerette protocol described in Devon et al., (1995) Nucleic Acids Research 23 (9): 1644-1645, with the modifications described by Stover (2001), UC Irvine (personal communication). Briefly, genomic DNA was digested with various restriction enzymes (Dra I, EcoR V, Pvu II, Sma I and Stu I for the Universal Genome Walker and BamH I method, EcoR I, Hind III and Xba I for the splinkerette method) and then it was ligated with adhesive end adapters for the Genome Walker method and with specific adapters of the restriction enzyme used for the splinkerette method. Adapters for both methods of genome-wide displacement were designed to prevent extension of the 3 'end of the adapter during PCR and thereby reduce or eliminate non-specific amplification. Fragments of genomic DNA linked to adapters were then referred to as libraries for genome-wide displacement or splinkerette libraries, one library per restriction enzyme. Libraries were prepared from isolated genomic DNA from three individual T1S2 plants with B.t. Cry34 / 35Abl of the event DAS-59122-7; plants DAS-59122-7 T1S2 1, DAS-59122-7 T1S2 2 and DAS-59122-7 T1S2 10, and a control plant Hi-II and a control plant PH09B.

After constructing the libraries, nested PCR amplifications were completed in order to amplify the target sequence using the Advantage ™ -GC Genomic PCR Kit (BD Biosciences Clontech, Palo Alto, CA). Primary PCR amplification employed a primer with identity for the adapter and a gene-specific primer. The adapter primer will not amplify a product in the first cycle of the primary PCR and only products will be produced from the gene-specific primer. Alignment at lower temperature and amplification from the primer to the adapter will only take place once the complementary strand is produced from the gene-specific primer. After amplification by primary PCR, a nested secondary PCR reaction was performed to increase the specificity of the genomic PCR reactions. The nested primers consisted of specific gene sequences and internal adapter specific for the respective primers used in the primary PCR. For the 5 'flanking sequences, the nested PCR was initiated using primers specific for the 5' end of the inserted T-DNA together with primers complementary to the adapter sequence linked to the digested DNA. Similarly, cloning of the 3 'flanking sequences started with a primer specific for the 3 'end of the inserted T-DNA and a primer complementary to the adapter sequence. Internal DNA sequences were used for the sequences of the right border and the left border of the T-DNA within the T-DNA region as starting points for the "shift" towards the maize genomic sequence, because they represented unique sequences (non-homologous). of the endogenous genomic sequences of corn) from which the primers could be anchored for displacement through the genome. The products produced by nested PCR were analyzed by agarose gel electrophoresis (data not shown). The fragments visible in the libraries prepared from one or more of the DNA samples of the event DAS-59122-7 and absent in the libraries prepared from the genomic DNA samples Hi-II and PH09B were identified for further characterization . The PCR-amplified fragments identified were separated by preparative gel electrophoresis, isolated using the QIAquick gel extraction element set (Qiagen) and sent directly for sequencing or cloned into the pGEM-T Easy plasmid vector using System I vectors pGEM-T Easy (Promega Corp., adison, WI) before DNA sequencing. The sequencing reactions were carried out with the primers used for nested PCR amplification or with the specific primers for use with the pGEM-T Easy vector. The sequence obtained was used to design other gene-specific primers to continue the "displacement" by the unknown genomic sequence of corn. Multiple rounds of displacement were applied through the genome until at least 500 bp of the edge sequence was obtained from the ends of the T-DNA insert. To ensure the validity of the sequences flanking the edges, additional amplifications were performed by event-specific PCR with genomic DNA from event DAS-59122-7. The amplified fragments were sequenced in order to further extend the region of superposition of the sequence of the region of the T-DNA insert with the genomic DNA of the 5 'and 3' borders. The primers, shown in Table 2, were designed on the basis of the sequence obtained from the genome-wide displacement experiments to amplify a fragment encompassing the unique binding of the T-DNA with the maize genomic DNA. Set of primers 03-O-506/02-O-476 (SEQ ID NO: 10 / SEQ ID NO: 9) spanned the 5 'junction and amplified a 313 bp fragment (from bp 2427 to bp 2739, see Figure 1) and set of primers 02-O-447/03-O-577 (SEQ ID NO: 8 / SEQ ID NO: 17) spanned the 3 'junction and amplified a fragment of 754 bp (from bp 9623 to bp 10376, see Figure 1).

Table 2. Sequences of the primers Location in the event sequence DASD59122D7 (see Figure 1). Bases 1 - 2593 = 5 'border, bases 2594 - 9936 = T-DNA insert, bases 9937 - 11922 = 3' border. To verify the DNA sequence that had been inserted into the maize genome, a PCR was performed to amplify, clone and sequence the inserted T-DNA of event DASD59122D7. The sets of primers for PCR were used (SEQ ID N °: 11 / SEQ ID N °: 5); (SEQ ID N °: 4 / SEQ ID N °: 7); and (SEQ ID N °: 6 / SEQ ID N °: 3) which are shown in Table 3 to amplify three overlapping fragments indicated as 221-1 (SEQ ID N °: 25), 221-2 (SEQ ID N °). : 26) and 221-3 (SEQ ID N °: 27) that represent the sequence of the 5 'region of the T-DNA that passes to the 3' region of the T-DNA insert from bp 2687 to bp 9846 for the event DAS-59122-7 (see Figure 1) . The information of the PCR amplicon is reported in Table 3 and the sequences of the primers are shown in Table 2.

Table 3. Descriptions of PCR primers and amplicons 1. Location in the event sequence DAS-59122-7 (see Figure 1). Bases 1 - 2593 = 5 'border, bases 2594 - 9936 = T-DNA insert, bases 9937 - 11922 = 3' border.

The GC2 Advantagea Polymerase PCR Kit (BD Biosciences Clontech, Inc.) was used according to the supplier's instructions to amplify the insert fragments (221-1 (SEQ ID N °: 25), 221-2 (SEQ ID N °: 26) and 221-3 (SEQ ID N °: 27)). Briefly, a reaction of 50 μ? contained 5 'and 3' primers at a final concentration of 0.2 μ? and 40 ng of genomic DNA. The PCR reactions were carried out in duplicate using the genomic DNA preparation of plants DAS-59122-7 T1S2 1 and DAS-59122-7 T1S2 2. The PCR conditions were: initial denaturation at 95 ° C for 1 min, followed of 35 cycles of 94 ° / 95 ° C for 30 sec, 55 ° C for 30 sec and 68 ° C for 5 min, with a final extension at 68 ° C for 6 min. The products of the PCR amplification were visualized under UV light, after electrophoresis through a 1% agarose gel in TBE IX (89 mM Tris-Borate, 2 mM EDTA, pH 8.3) stained with ethidium bromide. The PCR fragments 221-1 (SEQ ID NO: 25), 221-2 (SEQ ID NO: 26) and 221-3 (SEQ ID NO: 27) were purified by trimming the fragments of an agarose gel. , 8% in TBE IX stained with ethidium bromide and then the agarose fragments were purified using the QIAquick gel extraction element set (Qiagen). The PCR fragments were cloned into the pGEM-T Easy plasmid vector using the System I vector of pGEM-T Easy (Promega Corp.). The cloned fragments were verified with a minipreparation of the plasmid DNA (set of elements for QIAprep Spin Miniprep, Qiagen) and restriction digestion with Not I. Plasmid clones and / or PCR fragments purified from the insert were then sent for sequencing of the complete insert. Sequencing reactions were performed with the primers designed to be specific for the known T-DNA sequences or with specific primers for use with the pGEM-T Easy vector. All PCR primers were synthesized by Sigma-Genosys, Inc. (The Woodlands, TX), which were used at a final concentration of 0.2-0.4 μ? in PCR reactions. PCR reactions with isolated genomic DNA for Cry34 / 35Abl B.t. of events DASD59122D7, DASD45214D4 and DAS-45216-6, and of the unmodified control lines Hi-II and PH09B were used to confirm (1) the presence of maize genomic DNA in the regions of the sequenced edges of the DAS event. 59122-7, and (2) the specific amplification of the event by the junctions of the edges of the T-DNA insert and of the genomic DNA in event DAS-59122-7. The PCR primers designed to amplify the edge sequence flanking the insert in event DAS-59122-7 were used to confirm the presence of said regions in the unmodified control lines, as well as in event DAS-59122-7. Two (2) sets of primers were evaluated for each of the 5 'and 3' edges (four (4) games in total). The sets of primers 03-O-784/03-O-564 (SEQ ID N °: 18 / SEQ ID N °: 14) and 03-O-784/03-O-543 (SEQ ID N °: 18 / SEQ ID NO: 13) were used to amplify 136 bp and 263 bp fragments, respectively, of the 5 'border sequence with the T-DNA insert in the DAS-59122-7 event (Figures 2 and 3). Similarly, the sets of primers 03-O-569/03-O-577 (SEQ ID N °: 15 / SEQ ID N °: 17) and 03-O-570/03-O-542 (SEQ) were used. ID N °: 16 / SEQ ID N °: 12) to amplify fragments of 227 bp and 492 bp, respectively, of the 3 'genomic border (Figures 2 and 3). Primers designed to amplify the binding fragments of the T-DNA insert and sequence were used to establish event-specific PCR fragments for event DAS-59122-7. A set of primers was selected for each of the two junctions. The set of primers 03-O-78/02-O-215 (SEQ ID NO: 18 / SEQ ID NO: l) was designed to amplify a 555 bp fragment for the 5 'junction and set of primers 02 -O-219 / 03-0-577 (SEQ ID N °: 2 / SEQ ID N °: 17) was designed for the amplification of a 547 bp fragment for the 3 'junction. A set of primers, IVR1 (0197) (SEQ ID NO: 39) 5'-CCGCTGTATCACAAGGGCTGGTACC-3 'and IVR2 (0198) (SEQ ID NO: 40) 5'-GGAGCCCGTGTAGAGCATGACGATC-3', based on the endogenous corn invertase gene (Hurst et al., (1999) Molecular Breeding 5 (6): 579-586), to generate a 226 bp amplification product as an internal positive control for all maize genomic DNA samples. All primers for PCR were synthesized by Sigma-Genosys, Inc. and used at a final concentration of 0.2-0.4 μ? in PCR reactions. The sequences of the PCR primers are listed in Table 2. For PCR amplifications, the Advantage ™ -GC 2 PCR kit (BD Biosciences) was used according to the supplier's instructions. Approximately 10-100 ng of genomic DNA annealing was used for each 50 μm PCR reaction. . The PCR conditions were: initial denaturation of the annealing at 94 ° C for 5 min, followed by 35 cycles of 95 ° C for 1 minute, 60 ° C for 2 minutes and 72 ° C for 3 min, with a final extension to 72 ° C for 7 min. The products of the PCR amplification were visualized under UV light after electrophoresis on a% 1 agarose gel with TBE IX and ethidium bromide. The sequence data obtained for the T-DNA insert and the edge regions of event DAS-59122-7 were reviewed and assembled using the software Seqman II ™ Version 4.0.5 (DNAStar, Inc., Madison, I) . The sequences flanking the 5 'and 3' edges of the insert present in event DAS-59122-7 were used for the homology searches in the GenBank public databases with the In order to further characterize the insertion site in the maize genome. The analysis to identify the open reading frames in the junction regions between the flanking edges and the T-DNA insert in the DAS-59122-7 event was done using the NTI 8.0 vector (InforMax ™, Inc., Frederick, MD ). In total, 11922 bp of genomic DNA sequence from event DAS-59122-7 was confirmed (see Figure 1). At the 5 'end of the T-DNA insert, 2593 bp of the sequence flanking the border was identified and 1986 bp of the flanking sequence was obtained at the 3' end from fragments derived from the displacement experiments. for the genome. A total of 7160 bp of the T-DNA insert was cloned and sequenced using sets of PCR primers designed to amplify three overlapping fragments called 221-1 (2501 bp) (SEQ ID N °: 25), 221-2 (3027 bp) (SEQ ID N °: 26) and 221-3 (2830 bp) (SEQ ID N °: 27) which represent the sequence of the 5 'region of the T-DNA passing to the 3' region of the DNA insert- T for event DAS-59122-7 from bp 2687 to bp 9846 (see Figure 1). The remainder of the insert of the T-DNA region was sequenced from two PCR fragments, 0506/0476 (SEQ ID N °: 10 / SEQ ID N °: 9) and 0447/0577 (SEQ ID N °: 8) / SEQ ID NO: 17) covering the 5 'and 3' junctions, respectively, of the T-DNA insert with the maize genomic DNA. The Used primers were designed based on the sequence obtained with the genome-wide displacement experiments to amplify a fragment that spanned the unique binding of T-DNA with corn genomic DNA. Set of primers 03-O-506/03-O-476 (SEQ ID NO: 10 / SEQ ID NO: 9) encompassed the 5 'junction and allowed to amplify a fragment of 313 bp (from bp 2427 to bp 2739 ) and primers set 03O447 / 03-O-577 (SEQ ID NO: 8 / SEQ ID NO: 17) spanned the 3 'junction and allowed to amplify a fragment of 754 bp (from bp 9623 to bp 10376). Combined, a total of 7343 bp of the T-DNA insert was cloned in the DASD59122D7 event and sequenced (from bp 2594 to bp 9936, see Figure 1) and compared to the transformant plasmid sequence, PHP17662. Two nucleotide differences were observed at bp 6526 and bp 6562 in the region of the untranslated T-DNA insert of the wheat peroxidase promoter (see Figure 1). None of the base changes observed affected the composition of the open reading frame of the T-DNA insert. It was found that both regions of the 3 'and 5' ends of the T-DNA insert were intact, except for the deletion of the last 22 bp at the 5 'end and 25 bp at the 3' end encompassing the edge regions right and left of the T-DNA, respectively. Although it is known that T-DNA border sequences play a critical role in the insertion of T-DNA into the genome, this result is not unexpected given that inserts are often imperfect, particularly on the left border of T-DNA (Tinland (1996) Trends in Plant Science 1 (6): 178-184). The BLAST analysis (Basic Local Alignment Search Tool) of the genomic regions of the edges of event DAS-59122-7 showed a limited homology with the sequences available to the public (Reléase 138.0, GenBank , October 25, 2003). Analysis of the 5 'border region showed two areas with significant homology for the genomic and EST sequences (Sequence Marking Expressed [Expressed Sequence Tag]) of corn. The first area encompassed 179 bp (bp 477 at bp 655 of the edge sequence) and presents similarity to various molecular markers, chromosomal sequences and consensus sequences obtained by alignment of various ESTs. The second area is observed at bp 1080 to bp 1153 (74 bp) of the 5 'border sequence and is similar to numerous EST and genomic different corn sequences. The 3 'border region also exhibited two small non-contiguous regions of similarity to plant DNA sequences. The 3 'internal region of 162 bp (bp 9954 to bp 10115) showed similarity to the 3' untranslated end of two genomic alcohol dehydrogenase (adhl) genes from Zea mays as well as several consensus EST sequences. A smaller region (57 bp) in the middle of the 3 'edge (bp 10593 a bpl0649) showed similarity with the non-coding regions of multiple maize genomic sequences. In general, no homologous regions of more than 179 base pairs were identified in any of the genomic sequences of the borders, nor was more than one homologous region of the same known sequence found. Individual accesses that were similar to the edge sequences of event DAS-59122-7 were examined to determine if the insertion in event DAS-59122-7 was in the coding sequence of the characterized protein. None of the regions of similarity corresponded to coding sequences of any known protein. The local alignment of the entire sequence of the transformation plasmid, PHP17662, with the edge sequences of event DAS-59122-7 did not show significant homology, indicating that the regions of the flanking flanks of the T-DNA insert were not they contained fragments of the transformant plasmid. Therefore, the identification and characterization of the genomic sequence flanking the insertion site in event DAS-59122-7 were limited due to the absence of regions of significant homology with known sequences. It was analyzed in the 5 'and 3' binding regions between the corn genomic border sequence and the T-DNA insert in the event DAS-59122-7 the presence of new open reading. No open reading frames of significant size (> 100 amino acids) were identified in the binding regions of the 5 'or 3' ends (edges), indicating that no new open reading frames were generated as a result of the insertion of the T-DNA. In addition, the homology searches did not indicate the presence of open endogenous corn reading frames in the regions of the edges that could have been interrupted by the insertion of the T-DNA in Cry34 / 35Abl B.t of event DAS-59122-7. Example 5. Primers for PCR Primer pairs specific for the event DNA were used to produce a diagnostic amplicon for DAS-59122-7. These pairs of event primers include, by way of example, SEQ ID N °: 18 and SEQ ID N °: 1; SEQ ID N °: 2 and SEQ ID N °: 17; SEQ ID N °: 10 and SEQ ID N °: 9; and SEQ ID N °: 8 and SEQ ID N °: 17; and SEQ ID N °: 36 and SEQ ID N °: 37. In addition to these primer pairs, any pair of primers derived from SEQ ID N °: 21 and SEQ ID N °: 22 which, when used in a DNA amplification reaction produces a diagnostic DNA amplicon for or DAS-59122-7, constitutes an embodiment of the present invention. Any modification of these methods that employs DNA primers, or complements thereof, to produce an amplicon DNA molecule that is diagnostic for DAS-59122-7 is typical of art skill. Further, include pairs of control primers, comprising IVR1 (0197) / IVR2 (0198) (SEQ ID NO: 39 / SEQ ID NO: 40) for the amplification of an endogenous corn gene, as internal standards for the conditions of reaction. The analysis of plant tissue DNA extracts to verify the presence of event DAS-59122-7 should include a DNA control of positive tissue extract (a DNA sample known to contain the transgenic sequences). A successful amplification of the positive control shows that the PCR was run under conditions that allow the amplification of target sequences. A negative, or wild-type, control of extract DNA should also be included in which tempered DNA is provided either genomic DNA prepared from a non-transgenic plant or from a non-transgenic plant for DAS-59122-7. In addition, a negative control that does not contain temperate DNA from corn extract constitutes a useful estimate of the reagents and conditions used in the PCR protocol. Those skilled in the art of DNA amplification methods will be able to select other DNA primer molecules of sufficient length between SEQ ID NO: 21 and SEQ ID NO: 22, and will be able to optimize the conditions for the production of a DNA. Diagnostic amplicon for the event DAS-59122-7. The use of these sequences of DNA primers with modifications with respect to the methods that are shown in these Examples is within the scope of the invention. An amplicon in which there is at least one DNA primer molecule of sufficient length derived from SEQ ID NO: 21 and from SEQ ID NO: 22 which is diagnostic for event DAS-59122-7 constitutes an embodiment of the invention. An amplicon in which there is at least one DNA primer of sufficient length derived from any of the genetic elements of PHI17662A that is diagnostic for event DAS-59122-7, constitutes an embodiment of the invention. The assay for the DAS-59122-7 amplicon can be performed using a Stratagene Robocycler thermocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient, or by methods and apparatus known to those skilled in the art. Having illustrated and described the principles of the present invention, it will be apparent to those skilled in the art that it is possible to modify the invention in terms of layout and detail without departing from said principles. The authors claim all modifications that are within the spirit and scope of the appended claims. All publications and patent documents published in this specification are incorporated herein by way of reference to the same extent as if each individual patent application or publication was will incorporate specifically and individually as a reference.

Claims (49)

1. CLAIMS 1. An isolated DNA molecule, characterized in that it comprises a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence shown in SEQ ID No.:19; b) the nucleotide sequence shown in SEQ ID NO: 20; c) the nucleotide sequence shown in SEQ ID NO: 23; d) the nucleotide sequence shown in SEQ ID NO: 21; and e) the nucleotide sequence shown in SEQ ID NO: 22.
2. 2. A set of elements [kit] to identify the event DAS-59122-7 in a biological sample that allows detecting a specific region of DAS-59122 -7, characterized in that the set of elements comprises at least one first primer, which recognizes a sequence in SEQ ID N °: 19 or SEQ ID N °: 20.
3. 3. The set of elements of clause 2, characterized because it also comprises at least a second primer that recognizes a nucleotide sequence in a sequence selected from the group consisting of: a) the sequence of SEQ ID No.:24; Y b) the sequence of SEQ ID NO: 20.
4. 4. The set of elements of clause 2, characterized in that the first primer recognizes a sequence of nucleotides in a sequence selected from the group consisting of: a) the sequence of SEQ ID No.:19; and b) the sequence of SEQ ID NO: 20.
5. 5. The set of elements of clause 3, characterized in that the at least first and second primers, respectively, comprise a pair of sequences selected from the group consisting of: a) the sequences of SEQ ID N °: 18 and SEQ ID N °: l; b) the sequences of SEQ ID NO: 10 and SEQ ID NO: 9; c) the sequences of SEQ ID No. 2 and SEQ ID No. 17; d) the sequences of SEQ ID No. 8 and SEQ ID No. 17; and e) the sequences of SEQ ID No.:36 and SEQ ID No. 37.
6. 6. A set of elements for the detection of specific DNA for DNA binding of the event DAS-59122-7 of maize and its progeny, characterized because it comprises at least one DNA molecule of a sufficient length of contiguous DNA polynucleotides to function in a DNA detection method, which is homologous or complementary to a sequence selected from the group consisting of: a) the nucleotide sequence which is shown in SEQ ID NO: 21; Y b) the nucleotide sequence shown in SEQ ID NO: 22.
7. 7. A set of elements to identify event DAS-59122-7 in a biological sample, characterized in that the set of elements comprises a specific probe comprising a sequence that hybridizes with sequences selected from the group consisting of: a) the sequences of SEQ ID No.:19 and SEQ ID No.:24; and b) the sequences of SEQ ID No.:20 and SEQ ID No.:24; contiguous of it.
8. 8. A set of elements for the detection of DNA, characterized in that it comprises at least one DNA molecule of a sufficient length of contiguous nucleotides homologous or complementary to SEQ ID NO: 21 or SEQ ID NO: 22 that functions as a DNA primer or probe specific to the corn event DAS-59122-7 and the progeny thereof.
9. 9. A DNA construct, characterized in that it comprises: a first, second and third expression cassette, wherein said first expression cassette comprises, operatively linked: (a) a corn ubiquitin promoter; (b) a 5 'non-translated exon of a corn ubiquitin gene; (c) a first intron of corn ubiquitin; (d) a DNA molecule encoding Cry34Abl; Y (e) a Pinll transcription terminator; wherein said second expression cassette comprises, operatively linked: (i) a wheat peroxidase promoter; (ii) a DNA molecule encoding Cry35Abl; and (iii) a Pinll transcription terminator; and wherein the third expression cassette comprises, operatively linked: (1) 35S CaMV promoter; (2) a DNA molecule encoding pat; and (3) a 3 'transcription terminator of 35S (CaMV).
10. 10. A plant, characterized in that it comprises the DNA construction of clause 9.
11. 11. The plant of clause 10, characterized in that said plant is a corn plant.
12. 12. A method for identifying event DAS-59122-7 in a biological sample, characterized in that it comprises detecting a specific region of DAS-59122-7 with a first probe or primer that specifically recognizes a sequence in SEQ ID NO: 19 or in SEQ ID NO: 20.
13. 13. The method of clause 12, characterized in that it further comprises amplifying a DNA fragment from a nucleic acid present in the biological sample using a polymerase chain reaction with at least two primers, where the first primer recognizes a sequence in SEQ ID No.:19 or SEQ ID No.:20, and a second primer recognizes a sequence in SEQ ID No.:20 or SEQ ID No. 24.
14. 14. The method of clause 13, characterized because the first primer recognizes a sequence in SEQ ID No.:19 and the second primer recognizes a sequence in SEQ ID No.:24.
15. 15. The method of clause 13, characterized in that the first primer recognizes a sequence in the SEQ ID NO: 20 and a second primer recognizes a sequence in SEQ ID NO: 20.
16. 16. The method of clause 14, characterized in that the first and second primers comprise the sequence of SEQ ID No.:18 and of SEQ ID N °: 1, respectively.
17. 17. The method of clause 14, characterized in that the first and second primers comprise the sequence of the SEQ ID N °: 10 and of SEQ ID N °: 9, respectively.
18. 18. The method of clause 15, characterized in that the first and second primers comprise the sequence of SEQ ID No. 2 and SEQ ID No. 17, respectively.
19. 19. The method of clause 15, characterized in that the first and second primers comprise the sequence of SEQ ID NO: 8 and SEQ ID NO: 17, respectively.
20. 20. The method of clause 14, characterized in that the first and second primers comprise the sequence of SEQ ID No.:36 and SEQ ID No.: 37, respectively.
21. 21. The method of clause 16, characterized in that it comprises amplifying a fragment of about 555 bp using a PCR identification protocol of DAS-59122-7.
22. 22. The method of clause 17, characterized in that it comprises amplifying a fragment of approximately 313 bp using a PCR identification protocol of DAS-59122-7.
23. 23. The method of clause 18, characterized in that it comprises amplifying a fragment of approximately 547 bp using a PCR identification protocol of DAS-59122-7.
24. 24. The method of clause 19, characterized in that it comprises amplifying a fragment of approximately 754 bp using a PCR identification protocol of DAS-59122-7.
25. 25. The method of clause 20, characterized in that it comprises amplifying a fragment of approximately 104 bp using a PCR identification protocol of DAS-59122-7.
26. 26. A method for detecting the presence of the event DAS-59122-7 of maize or progeny thereof in a biological sample, characterized in that it comprises: (a) extracting a DNA sample from said biological sample; (b) providing a pair of DNA primer molecules selected from the group consisting of: i) the sequences of SEQ ID No.:18 and SEQ ID No.:1; ii) the sequences of SEQ ID NO: 10 and SEQ ID NO: 9; iii) the sequences of SEQ ID No. 2 and SEQ ID No. 17; Y iv) the sequences of SEQ ID No. 8 and SEQ ID No. 17; (c) providing the conditions for a DNA amplification reaction; (d) performing the DNA amplification reaction, thereby producing a DNA amplicon molecule; Y (e) detecting the DNA amplicon molecule, where the detection of said DNA amplicon molecule in the DNA amplification reaction indicates the presence of the corn event DAS-59122-7.
27. 27. An isolated DNA molecule, characterized in that it comprises any of the amplicons produced by the method of clause 26.
28. 28. A method for detecting the presence of DNA corresponding to the event DAS-59122-7 in a sample, characterized in that the method comprises: (a) putting the sample comprising corn DNA in contact with a polynucleotide probe that hybridizes under severe hybridization conditions with DNA from the corn event DAS-59122-7 and does not hybridize under such severe hybridization conditions with a plant DNA that is of DAS-59122-7 corn; (b) subjecting the sample and the probe to severe hybridization conditions; and (c) detecting the hybridization of the probe with the DNA, where the detection of the hybridization indicates the presence of the event DAS-59122-7.
29. 29. A DNA nucleotide sequence isolated from a primer, characterized in that it comprises a sequence selected from the group consisting of: SEQ ID N °: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 36 and 37, or a complement thereof.
30. 30. The nucleotide sequence of DNA isolated from a primer of clause 29, characterized in that it comprises a sequence selected from the group consisting of: SEQ ID No .: 1, 2, 8, 9, 10, 17 and 18, or a complement of it.
31. 31. A pair of DNA molecules, characterized in that it comprises: a first DNA molecule and a second DNA molecule, wherein the DNA molecules are of a sufficient length of contiguous nucleotides of a sequence selected from the group consisting of: a) the sequence shown in SEQ ID N °: 21 or a complement thereof; and b) the sequence shown in SEQ ID No.:22 or a complement thereto; to function as primers or diagnostic DNA probes for the DNA extracted from a maize plant DAS-59122-7 or progeny thereof.
32. 32. An isolated DNA molecule, characterized in that it comprises a binding sequence comprising a sequence selected from the group formed by SEQ ID N °: 32, 33, 34 and 35 and complements thereof.
33. 33. A method for confirming seed purity, characterized in that it comprises detecting a specific region of DAS-59122-7 with a specific primer or probe that specifically recognizes a sequence in SEQ ID No.:19 or SEQ ID No. : 20, in a sample of seeds.
34. 34. A method for screening seeds for the presence of event DAS-59122-7, characterized in that it comprises detecting a specific region of DAS-59122-7 with a specific primer or probe that specifically recognizes a sequence in SEQ ID NO: 19 or in SEQ ID N °: 20 in a sample of a seed lot.
35. 35. An insect resistant maize plant, or parts thereof, characterized in that it contains a DNA having at least one nucleotide sequence selected from the group consisting of SEQ ID N °: 32, 33, 34 and 35, and complements of them, are part of the genome of the plant.
36. 36. A plant that descends from the insect resistant maize plant of clause 35, characterized in that it contains DNA having at least one nucleotide sequence selected from the group consisting of SEQ ID N °: 32, 33, 34 and 35, and complements thereof, is part of the genome of the plant.
37. 37. Seeds, characterized because they are from the plant of clause 35 or 36.
38. 38. A method for producing an insect-resistant corn plant, characterized in that it comprises the planting of a plant of clause 35 or 36 and the selection of the progeny by analyzing the presence of at least one nucleotide sequence selected from the group formed by SEQ ID N °: 32, 33, 34 and 35 and complements thereof.
39. 39. An isolated DNA sequence, characterized in that it comprises at least one nucleotide sequence selected from the group consisting of SEQ ID N °: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 36 and 37, or complements thereof.
40. 40. A pair of isolated DNA sequences, characterized in that each of them comprises at least ten nucleotides and that when used together in a DNA amplification procedure will produce a diagnostic amplicon for event DAS-59122-7.
41. 41. The pair of DNA sequences isolated from clause 40, characterized in that each sequence is selected from a nucleotide sequence selected from the group consisting of: a) the sequence of SEQ ID No.:21; and b) the sequence of SEQ ID NO: 22.
42. 42. A method for detecting the presence of the insertion of event DAS-59122-7 in corn tissue, characterized in that it comprises: (a) selecting a pair of primers, each of which comprises at least ten nucleotides of SEQ ID NO: 21 or SEQ ID NO: 22 where each member of the pair is on opposite sides of a diagnostic sequence for said event insertion DAS-59122-7; (b) putting the sample of said corn tissue in contact with said pair of primers; (c) perform a DNA amplification and analyze the amplicons.
43. 43. The method of clause 42, characterized in that the pair of primers is selected from the group consisting of SEQ ID N °: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 36 and 37 or complements thereof.
44. 44. A method for detecting the presence of the insertion of event DAS-59122-7 in corn tissue, characterized in that it comprises: (a) putting a sample of said corn tissue in contact with a polynucleotide probe that hybridizes under conditions of severe hybridization with one or more DNA sequences selected from the group formed by SEQ ID N °: 32, 33, 34 and 35 and complements thereof; (b) subjecting the sample and probe to severe hybridization conditions; and (c) analyzing the hybridization of the probe.
45. 45. A set of elements for the detection of DNA, characterized in that it comprises a polynucleotide probe that hybridizes under severe hybridization conditions with one or more DNA sequences selected from the group formed by SEQ ID NO: 32, 33, 34 and 35 and complements thereof.
46. 46. A set of elements for the detection of DNA, characterized in that it comprises a pair of primers each of which comprises at least 10 nucleotides of SEQ ID N °: 21 and SEQ ID N °: 22, where each one it is on opposite sides of a diagnostic sequence for the insertion of event DAS-59122-7.
47. 47. The DNA detection set of elements of clause 46, characterized in that the pair of primers is selected from the group consisting of SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 36 and 37 and complements thereof.
48. 48. A set of elements to identify event DAS-59122-7 in a biological sample, characterized in that it allows detecting a specific region of DAS-59122-7 in SEQ ID NO: 23.
49. 49. A method to identify DAS- 59122-7 in a biological sample, characterized in that it allows detecting a specific region of DAS-59122-7 in SEQ ID NO: 23.