RU2577143C2 - Detection of aad-1 of object das-40278-9 - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method for determining zygosity of corn plant, containing transgenic construction, which contains arylozyalkanoate dioxigenase, where said method includes: obtaining sample of genome DNA from said corn plant; obtaining sample, brought in contact, by contact of said DNA sample with first primer and second primer and fluorescent probe, determination of zygosity of said corn plant, as well as to set for its realisation.

EFFECT: invention makes it possible to effectively determine zygosity of corn plant, containing arylozyalkanoate dioxigenase gene.

15 cl, 3 dwg, 1 tbl, 1 ex

Description

BACKGROUND OF THE INVENTION

The aad -1 gene (originally the Sphingobium herbicidovorans gene) encodes an aryloxyalkanoate dioxygenase (AAD-1) protein. This feature provides resistance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate herbicides (which are commonly referred to as “fop” herbicides such as diclofop and kisalofop) and can be used as a selective marker during plant transformation, as well as in breeding nurseries. Role aad -1 gene for herbicide resistance first disclosed in WO 2005/107437 (see. Also US 2009-0093366).

There are various methods for detecting an object (event). However, they all have their drawbacks. One method is pyrosequencing as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). According to this method, an oligonucleotide is constructed that overlaps with the junction of the flanking genomic DNA and the DNA insert. The oligonucleotide is hybridized with a single-stranded PCR product from the desired region (with one primer in the inserted sequence and one primer in the flanking genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5'-phosphosulfate and luciferin. Separately, DNTP is added and the inclusion results are evaluated by the light signal. Upon successful amplification, hybridization, and extension to one or multiple bases, the signal indicates the presence of an insert / flanking sequence.

Fluorescence polarization is another method that can be used to detect the amplicon of the present invention. According to this method, an oligonucleotide is constructed that overlaps with the junction of the genomic flanking sequence and the DNA insert. The oligonucleotide is hybridized with a single-stranded PCR product from the desired region (with one primer in the inserted sequence and one primer in the flanking genomic sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTP. Single nucleotide extension leads to the inclusion of ddNTP. Inclusion can be estimated by the change in polarization using a fluorimeter. Upon successful amplification, hybridization, and one-base extension, a change in polarization indicates the presence of a transgenic insert / flanking sequence.

Molecular signal indicators for use in sequence detection are described. In summary, an FRET oligonucleotide probe is constructed that overlaps with the junction of the genomic flanking sequence and the DNA insert. Due to its unique structure, the FRET probe contains a secondary structure that holds the fluorescent and quenching fragments in close proximity. The FRET probe and PCR primers (one primer in the inserted sequence and one primer in the flanking genomic sequence) are subjected to hybridization cycles in the presence of thermostable polymerase and dNTP. After successful PCR amplification, hybridization of the FRET probe with the target sequence leads to the removal of the secondary structure of the probe and the spatial separation of the fluorescent part from the non-fluorescent part. As a result, a fluorescent signal is produced. Upon successful amplification and hybridization, this fluorescent signal indicates the presence of a flanking sequence / transgenic insert.

Probe hydrolysis analysis, otherwise known as the TAQMAN method (PE Applied Biosystems, Foster City, California), is a method for detecting and quantifying the presence of a DNA sequence. In summary, an FRET oligonucleotide probe is constructed that overlaps with the junction of the genomic flanking sequence and the DNA insert. The FRET probe and PCR primers (one primer in the inserted DNA and one primer in the flanking genomic DNA sequence) are subjected to hybridization cycles in the presence of thermostable polymerase and dNTP. Hybridization of the FRET probe results in cleavage and release of the fluorescent portion from the non-fluorescent portion on the FRET probe. Upon successful amplification, hybridization, and extension to one base, a fluorescent signal indicates the presence of a flanking sequence / transgenic insert.

Another challenge, among many others, is to find a suitable reference gene for a given test. For example, as cited in an abstract by Czechowski et al., "An exceptionally large group of data from the Affymetrix ATH1 GeneChip genome analysis provides methods for identifying a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis { Arabidopsis thaliana ). Hundreds of genes have been found. "Arabidopsis superior to traditionally used reference genes with respect to expression stability during development and under various environmental conditions." (Czechowski et al. (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5-17.)

Brodmann et al. (2002) describes real-time quantitative PCR for detecting transgenic corn in food for four different varieties of corn approved by the European Union.

Brodmann, P.D., P.D., Ilg E. C, Berthoud H., and Herrmann, A. Real-Time Quantitative Polymerase Chain Reaction Methods for Four Genetically Modified Maize Varieties and Maize DNA Content in Food. J. of AOAC international 2002 85 (3).

Hernandez et al. (2004) indicates four possible genes for use in real-time PCR.

Hernandez, M., Duplan, M.-N., Berthier, G., Vaitilingom, M., Hauser, W., Freyer, R., Pla, M., and Bertheau, Y. Development and comparison of four real- time polymerase chain reaction systems for specific detection and quantification of Zea mays LJ Agric. Food Chem. 2004, 52, 4632-4637.

Costa et al. (2007) studied these four genes (also in the context of real-time PCR) and concluded that alcohol dehydrogenase and zein genes are the best reference genes for detecting “object” samples (lectin gene) in transgenic food products. Costa, L. D., and Martinelli L. Development of a Real-Time PCR Method Based on Duplo Target Plasmids for Determining an Unexpected Genetically Modified Soybean Intermix with Feed Components. J. Agric. Food Chem. 2007, 55, 1264-1273.

Huang et al. (2004) used plasmids pMulM2 as reference molecules for the detection of MON810 and NK603 transgenes in maize. Huang and Pan, "Detection of Genetically Modified Maize MON810 and NK603 by Multiplex and Real-Time Polymerase Chain Reaction Methods," J. Agric. Food Chem., 2004, 52 (11), pp. 3264-3268.

Gasparic et al. (2008) proposed ZNK technology based on the method of probe cyclization, TaqMan, and various real-time PCR components for quantifying corn objects (such as MON810). Gasparic, Cankar, ZeI, and Gruden, "Comparison of different realtime PCR chemistries and their suitability for detection and quantification of genetically modified organisms," BMC Biotechnol. 2008; 8: 26.

US 20070148646 relates to a quantitative evaluation primer extension method that requires the controlled release of individual nucleotides that can be detected and quantified based on the number of nucleotides incorporated. This method differs from the TaqMan PCR method, in which a whole reference gene is used.

To distinguish between homozygous and hemizygous genotypes, TC 1507 successfully performed Invader analysis for this object. Gupta, M., Nirunsuksiri, W., Schulenberg, G., Hartl, T., Novak, S., Bryan, J., Vanopdorp, N., Bing, J. and Thompson, S. A non-PCR-based Invader Assay Quantitatively Detects Single-Copy Genes in Complex Plant Genomes. MoI Breeding 2008, 21, 173-181.

Huabang (2009) describes a PCR-based zygosity analysis of transgenic maize. However, reference genes were not used in this work. Huabang, "An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically Modified Maize," Molecular Plant Breeding, 2009, Vol. 7, No.3, 619-623.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence of an AAD-1 corn object, designated DAS-40278-9, in a sample (e.g., corn grain). (Samples of seeds deposited with the American Type Culture Collection (ATCC) under accession number PTA-10244 (hybrid yellow dent corn seeds (Zea Mays L):. DAS -40278-9; deposited under the Budapest Treaty Dow AgroSciences LLC; date of receipt of seeds / strain (s) at ATTC: July 10, 2009; viability confirmed August 17, 2009.) Also sets and conditions used in the analysis are given.

More specifically, the present invention relates in part to TaqMan end-point PCR analysis for a maize AAD-1 object using endogenous maize reference genes. Some embodiments are directed to high throughput zygosity analysis methods. The present invention further relates in part to the search for preferred invertase reference genes for use in determining zygosity.

Thus, this invention also partially relates to plant breeding comprising the detection methods described herein. In some embodiments, said object can be “combined” with other traits, including, for example, other herbicide resistance gene (s) and / or insect resistance proteins. These procedures can be used to uniquely identify corn lines containing an object of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 shows a strategy for cloning a DNA insert into a corn object DAS-40278-9.

In FIG. Figure 2 shows the scheme of the primers used in PCR amplification to confirm the presence of flanking border regions in the DAS-40278-9 object of maize. The scheme displays the location of the primers to confirm the full-length sequence of the DAS-40278-9 object AAD-1 corn from the 5'-end to 3'-end.

In FIG. 3 illustrates a cloning strategy for the flanking border regions of a corn object DAS-40278-9 The genomic DNA of the corn object DAS-40278-9 was digested with Eco R V, Stu I or Sca I and the corresponding GenomeWalker ™ libraries were compiled, which were used as a template for amplification of the desired DNA sequence.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is a sequence of 5 ′ and 3 ′ flanking genomic sequences on each side of an AAD-1 insert, including the insert, for maize DAS-40278-9.

SEQ ID NO: 2-7 are primers and probes for use in the present invention.

SEQ ID NO: 8 is an example of an amplicon of an object.

SEQ ID NO: 9 is an example of a reference amplicon.

DETAILED DESCRIPTION OF THE INVENTION

The transgenic object DAS-40278-9 of corn AAD-1 (providing resistance to herbicides) was obtained by transformation by shaking cells in a suspension of microneedles. The flanking sequences of both the 5 ′ end and the 3 ′ end of this AAD-1 transgenic insert were cloned, sequenced, and analyzed as described in USSN 61/235248 (filed August 19, 2009).

Special TAQMAN primers and probes were designed as described herein, partially corresponding to the DNA sequences located at the 5'-end of the insert and genomic DNA compound. A successful duplex analysis of the object-specificity of primers and probes was performed by real-time PCR for 16 different maize AAD-1 objects and two non-transgenic maize varieties with the maize invertase gene as a reference gene. Procedures have been developed for processing the results of an object-specific analysis of TAQMAN for DAS-40278-9 AAD-1 maize, as described herein.

The sequence, including the junction of the host plant DNA and the integrated gene construct in this AAD-1 corn, is unique. It was used to conduct object-specific analyzes (traditional real-time PCR or PCR) to detect the presence of AAD-1 maize DAS-40278-9 for testing GMOs and determining the zygosity of plants in a breeding population. The TAQMAN object-specific analysis described herein can be used for both purposes.

The present invention provides assays to detect the presence of a corn transgenic object DAS-40278-9 of interest (also known as pDAS 1740-278) in a sample. Aspects of the present invention include methods for constructing and / or producing diagnostic nucleic acid molecules, exemplified or provided herein.

The present invention also partially relates to plant breeding, including any of these methods. In some embodiments, an object of the present invention may be “combined” with other traits (for example, such as other herbicide resistance gene (s) and / or genes (s) encoding insect resistance proteins). Plant lines containing the indicated object can be detected using the sequences disclosed and provided herein.

In some embodiments, the present invention relates to the identification of herbicide-resistant maize lines. The present invention relates in part to detecting the presence of said object to determine whether the descendants of the cross carry an object of interest. In addition, a method for detecting an object is provided, useful, for example, from the point of view of complying with the rules prescribed by regulatory authorities that give permission for a given product to enter the market and require, for example, the presence on the packaging of a food product of information that it was obtained from recombinant agricultural plants.

The present invention partially relates to fluorescence-based processing of TaqMan PCR analysis results using an endogenous gene as a reference (number of copies) controls for analyzing the zygosity of an AAD-1 maize object with high throughput. The present invention relates in part to the discovery of a preferred reference gene, invertase. Several reference genes are indicated as possible.

The present invention also partially relates to the development of an TaqMan endpoint PCR assay for an AAD-1 object-specific zygosity assay. Further, the present invention partially relates to the production of test kits for the selection of AAD-1.

Endpoint TaqMan assays are based on a plus or minus strategy, where plus indicates a sample positive for the gene being studied, and minus indicates a sample negative for the studied gene. These assays typically use two sets of oligonucleotides to determine the AAD-1 transgenic sequence and the wild-type gene sequence, respectively, as well as double-labeled probes to measure the content of the transgenic sequence and the wild-type sequence.

Although Invader analysis is recognized as a reliable way to evaluate objects, it is very sensitive to DNA quality. In addition, large amounts of DNA are required for analysis. An Invader analysis also requires an additional denaturation step, if not performed correctly, Invader analysis may be unsuccessful. In addition, Invader analysis takes a lot of time, which does not allow efficient processing of a large number of AAD-1 samples for analysis for commercial purposes. The main advantage of the present invention is to reduce analysis time and to eliminate the denaturation step.

The end-point TaqMan analysis under consideration for detecting AAD-1 objects provides unexpected advantages over Invader analysis, especially when processing a large number of samples.

The present invention can help to obtain and describe signs of resistance of AAD-1 to herbicides in crops, including corn, soy and cotton.

Definitions and examples are provided herein for the convenience of describing the present invention and for indicating to those skilled in the art how to practice the invention. Unless otherwise indicated, the terms should be understood according to traditional use by those skilled in the art. The DNA base designation system given in 37 CFR §1.822 was used. As used herein, the term "offspring" refers to the descendants of any generation of the parent plant that contains the AAD-1 maize object DAS-40278-9.

A transgenic “event” is obtained by transforming plant cells with heterologous DNA, i.e. a nucleic acid construct that includes the necessary transgene, followed by regeneration of the plant population by incorporating the transgene into the plant genome and selecting a particular plant, characterized by the presence of an insert at a particular position of the genome. The term "object" refers to the original transformant and offspring of this transformant, which includes heterologous DNA. The term "object" also means offspring produced by sexual outcrossing between a given transformant and another variety that includes heterologous DNA. Even after repeated crossbreeding with the recurrent parent, the integrated DNA and flanking DNA from the transformed parent are present in the offspring of this cross in the same chromosome position. The term “object” also means DNA from a source object containing an integrated DNA and a flanking genomic sequence directly adjacent to the integrated DNA, which is expected to be transmitted to offspring that inherits the integrated DNA including the desired transgene transmitted as a result of sexual crosses of the parent line containing embedded DNA (for example, the original transformant and offspring resulting from self-pollination), with a parent line that does not contain embedded DNA.

The “junction sequence" encompasses the region in which the DNA embedded in the genome mates with the DNA of the natural maize genome flanking the insertion site. It includes DNA sequences spanning the inserts in the corn objects described herein and having the same length as the flanking DNAs.

The present invention relates to the identification of the desired object. Appropriate PCR primers and amplicons are included in the invention. These molecules can be used to detect or determine commercial varieties of transgenic corn or lines derived from the transgenic corn lines in question.

The complete insertion sequence along with portions of the corresponding flanking sequences is presented herein in SEQ ID NO: 1. The sites of the insertion and flanking sequences for this object with respect to SEQ ID NO: 1 (total 8557 nucleotides) are given below.

The components of the AAD-1 insert and flanking sequences for this object are further illustrated in FIG. 1-3.

The detection methods of the present invention can be used in combination with plant breeding to determine which offspring contain this object after crossing the parent plant containing the object of interest with another line plant in order to introduce one or more additional traits of interest into the offspring. The methods of the invention can be used, for example, in corn breeding programs, as well as in quality control, especially for transgenic corn seeds used for commercial purposes. It can also facilitate product registration and production management. These methods can be used for improved selection strategies.

In some embodiments, the implementation of fluorescence-based TaqMan endpoint analysis for determining zygosity allows direct analysis of the results in a spectrophotometer for reading tablets with identification of the AAD-1 object in corn and the reference gene.

The present invention includes applications in breeding, such as analysis of the introgression of an AAD-1 object into other maize lines.

Detection methods and kits of the present invention can be used to identify objects according to the present invention. The methods and kits of the present invention can be used for improved strategies for selection and analysis of gene linkage.

The detection methods of the present invention are particularly effective in combination with plant breeding to determine which offspring contain the given object after crossing the parent plant containing the object of interest with another line plant in order to transmit one or more additional traits of interest to the offspring. These Taqman PCR assay methods are effective for use in maize breeding programs as well as in quality control, especially for commercially used maize seeds. Taqman PCR kits can also be prepared and used for such transgenic maize lines. It can also be effective for product registration and production management.

Moreover, these methods can be used to study and describe the process of transgene integration, to describe the genomic integration site, sort objects, stability of transgenes and their flanking sequences and gene expression (especially related to suppression of gene transcription, transgene methylation patterns, position effect and potentially related expression by elements such as MARS [regions of attachment to the nuclear matrix], etc.).

As used herein, the term "corn" means maize ( Zea mays ) and includes all varieties that can be crossed with corn.

The present invention further includes crossing methods and the application of the methods of the present invention. For example, the present invention includes a method for producing F ₁ hybrid seeds by crossing an exemplary plant with another (eg, inbred parent) plant to produce hybrid seeds, and detecting the desired object. The qualities of the resulting plants can also be improved by combining the methods of the present invention.

Herbicide-resistant corn plants can be crossed by sexually mating the first parent plant, which is a corn plant grown from the seeds of the line described herein, with a second parent corn plant to produce multiple first-generation plants; and then selecting a first-generation plant resistant to the herbicide (or carrying the subject in question); and self-pollination of plants of the first generation to obtain many plants of the second generation; and then selection from plants of the second generation of the plant resistant to the herbicide (or carrying at least one of the objects). These steps may further include backcrossing the first generation plant or second generation plant with the second parent corn plant or third parent corn plant. A grain crop containing the corn seeds of the present invention, or their offspring, can be planted.

It should also be understood that two different transgenic plants can also be crossed to produce offspring that contain two independently segregated exogenous genes. As a result of self-pollination of the corresponding offspring, it is possible to obtain plants homozygous for both of these exogenous genes. The present invention also contemplates crossbreeding with a parent plant and outcrossing with a non-transgenic plant, as well as vegetative propagation. Other crossing methods commonly used for various traits and cultures are also known in the art. Backcrossing was used to transfer genes encoding traits with simple dominant inheritance to the desired homozygous cultural or inbred line, which is a recurrent parent. The carrier plant of the trait to be transferred is called the donor parent. The resulting plant is expected to bear the traits of a recursive parent (e.g., cultivar) and the desired trait received from the donor parent. After the first crossover, plants possessing the phenotype of the donor parent are selected, and they cross-cross (backcrossing) with the recurrent parent. The resulting plant is expected to bear the traits of a recursive parent (e.g., cultivar) and the desired trait received from the donor parent.

The present invention can be used in combination with a marker crossing method (MAB). Also, the DNA molecules of the present invention can be used with other methods (such as AFLP markers, RFLP markers, RAPD, SNP and SSR markers), which allow the identification of genetically linked agronomically useful traits. Signs of resistance to herbicides can be monitored in the offspring of cross (or offspring of this offspring and any other cultivars or varieties of corn) using MAB methods. The methods of the present invention can be used to identify any varieties of maize bearing an object of interest.

The methods of the present invention include a method for producing herbicide-resistant maize plants, wherein said method comprises crossbreeding with a plant bearing an object of interest. Preferred methods further include selecting offspring from said cross by checking said offspring for the presence of an object detectable according to the present invention. For example, the present invention can be used to track a desired object in cross-breeding cycles of plants bearing desired traits, such as agronomic traits. Plants containing the desired object and desired traits can, for example, be detected, identified, selected and quickly applied in further crosses. The desired object / sign can also be combined by crossing with the sign (s) of resistance to insects and / or additional signs of resistance to herbicides and track according to the present invention. One preferred embodiment is a plant containing the desired object in combination with a gene encoding the herbicide resistance of imidazolinone, glyphosate and / or glufosinate. In some embodiments, a dicamba resistance gene can be used.

Thus, the present invention can be combined with, for example, genes encoding glyphosate resistance (e.g., a resistant plant or bacterial EPSPS, GOX, GAT), glufosinate resistance (e.g. Pat, bar), resistance to acetolactate synthase inhibiting herbicides (ALS ) (for example, imidazolinones [such as imazetapyr], sulfonylcarbamide, triazolopyrimidine sulfonanilide, pyrimidinylthiobenzoates and other chemical compounds [Csrl, SurA, et al.]), resistance to bromoxynil (eg Bxn), resistance to enzyme inhibitors 4 (enzyme 4) hydroxyphenyl pyruvate dioxygenase), resistance to phytoene desaturase inhibitors (PDS), resistance to photosystem II inhibitors (e.g. psbA), resistance to photosystem I inhibitors, resistance to protoporphyrinogen oxidase IX inhibitors (PPO) (e.g. , PPO-I), resistance to phenyluric herbicides (e.g., CYP76B1), dicamba-degrading enzymes (see, for example, US 20030135879), and others that can be combined individually or in complex combinations to provide effective control or other dotvrascheniya infection weeds and / or herbicide resistance classes mentioned above.

For additional herbicides, some additional preferred ALS (also known as AHAS) inhibitors include triazolopyrimidine sulfonanilides (such as cloransulam methyl, diclosulam, flurasulam, flumetsulam, metosulam and penoxulam), pyrimidinyl thiobenzoates (such as bispiribibacone and). Some preferred HPPD inhibitors include mesotrione, isoxaflutol, and sulcotrione. Some preferred PPO inhibitors include flumiclorac, flumioxazine, flufenpyr, piraflufen, flutiacet, butafenacil, carfentrazone, sulfentrazone and diphenylethers (such as acifluorfen, fomesafen, lactofen and oxyfluorfen).

In addition, AAD-1, individually or in combination with one or more additional HTC features, can be combined with one or more additional primary (for example, insect resistance, resistance to fungi or stress resistance, etc.) or secondary (for example, increased productivity, improved characteristics of oils, increased quality of fibers, etc.) signs. Thus, the present invention can be applied to provide a complete agronomic set, including improved crop quality and the ability to flexibly control any number of agricultural pests with optimal costs.

As used herein, a “line” is a group of plants that is characterized by low genetic diversity or lack thereof between individuals for at least one trait. Such lines can be obtained by self-pollination and selection for several successive generations, or by vegetative propagation of one parent plant using tissue or cell culture methods.

As used herein, the terms "cultivar" and "variety" are synonymous and refer to the line used for commercial production.

"Stability" or "stable" means that in relation to this component, the component is obtained from generation to generation, and preferably at least for three generations, at almost the same level, for example, preferably ± 15%, more preferably ± 10 %, most preferably ± 5%. Stability can be affected by temperature, location, stress, and landing time. When comparing successive generations in the field, the levels of component production should be similar.

By "commercial suitability" is meant a high plant capacity and high fertility, in connection with which farmers using traditional farm equipment can get a crop, and oil with the described components can be extracted from seeds using traditional crushing and extraction equipment. A commercially viable crop, when estimated by seed weight, oil content and total oil per acre, differs by no more than 15% from the average yield of a comparable commercial variety of maize without signs of exceptional growth in the same region.

“Agronomically valuable” means that the line possesses the desired agronomic characteristics, such as yield, ripeness, disease resistance, etc., in addition to insect resistance due to the object (s) in question. Agronomic traits are considered individually or in combination.

Specialists in this field in the light of the present invention should understand that the preferred embodiments of the kits for detection, for example, may include probes and / or primers. For example, they include polynucleotide probes, primers, and / or amplicons, as described herein.

Specialists in this field should also understand that it is possible to design primers and probes for hybridization in the range of standard hybridization conditions and / or PCR with the plot of SEQ ID NO: 1 (or a complementary sequence) and its complementary sequences, where the primer or probe is not completely complementary to the above as an example of a sequence. Thus, a degree of complementarity violation can be tolerated. For example, for a primer of approximately 20 nucleotides in length, typically one or two or the like. nucleotides may not bind to the opposite strand if the non-complementary base is internal or is located at the end of the primer opposite the amplicon. Various suitable hybridization conditions are given below. Synthetic nucleotide analogs, such as inosine, can also be used in probes. You can also use probes from peptide nucleic acids (PNAs), as well as DNA and RNA probes. It is important that such probes and primers are diagnostic features (capable of uniquely identifying and distinguishing) for the presence of the object of the present invention.

The components of each insert are shown in FIG. 1-3. Polynucleotide DNA sequences of these components or fragments thereof can be used as DNA primers or probes in the methods of the present invention.

In some embodiments, compositions and methods are provided for detecting the presence of a transgene / genome insertion region in plants and seeds and the like. corn.

In some embodiments, DNA sequences containing an adjacent fragment of a new transgene / genome insertion region are aspects of the present invention. DNA sequences are included that contain a sufficient amount of polynucleotides from the transgenic insert sequence and a sufficient number of polynucleotides from the genomic sequence of the maize and / or the sequence used as a primer sequence to produce an amplicon for the diagnosis of one or more such maize plants.

Related embodiments relate to DNA sequences containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides of the transgenic portion of the DNA sequence described herein, or its complementary sequences. Such sequences can be used as DNA primers in DNA amplification methods. Amplicons obtained using these primers are diagnostic indications of the presence of the corn object indicated in this document. Thus, the invention also includes amplicons obtained using such DNA primers and homologous primers.

The present invention includes methods for detecting the presence in the sample of DNA corresponding to the DNA of the corn object specified herein. Such methods may include the steps of: (a) contacting the biological sample with a pair of primers which, when used in the amplification reaction of a nucleic acid with said DNA, produce an amplicon, which is a diagnostic indicator of the presence of said object (s); (b) conducting a nucleic acid amplification reaction to produce an amplicon; and (c) detecting said amplicon.

Further detection methods of the present invention include a method for detecting the presence of DNA in a sample corresponding to at least one of said objects, wherein said method comprises the steps of: (a) contacting a biological sample with a probe that hybridizes under stringent hybridization conditions with said DNA and does not hybridize under stringent hybridization conditions with the DNA of a control corn plant (containing no embedded DNA of interest); (b) creating stringent hybridization conditions for said sample and probe; and (c) detecting hybridization of said probe with DNA.

The present invention includes methods for detecting the presence of DNA in a sample from at least one corn plant specified herein. Such methods may include the steps of: (a) contacting the biological sample with a pair of primers which, when used in the amplification reaction of a nucleic acid with said DNA, produce an amplicon, which is a diagnostic indicator of the presence of the indicated object (s); (b) carrying out a TAQMAN PCR amplification reaction using the reference gene specified herein; and (c) analysis of the results.

In further embodiments, the present invention includes methods for producing a corn plant containing the AAD-1 subject of the present invention, wherein said method comprises the steps of: (a) sexually crossing the first parent line of the corn (containing expression cassettes of the present invention that provide said sign of resistance to herbicides to plants of the indicated line) and the second parent line of maize (devoid of the characteristic of resistance to herbicides) to obtain many descendant plants; and (b) selection of offspring using molecular markers. Such methods optionally comprise a further step of backcrossing the offspring with the second parent line of the corn to produce a homozygous plant bearing the indicated herbicide resistance trait.

In another aspect of the invention, methods are provided for determining the zygosity of offspring of a cross using the subject in question. These methods may include contacting a sample containing corn DNA with a pair of primers of the present invention. These primers, when used in a nucleic acid amplification reaction with said DNA, produce the first amplicon, which is a diagnostic sign of the presence of at least one of these corn objects. Such methods further include carrying out a nucleic acid amplification reaction to produce a first amplicon; detection of the first amplicon; and contacting a sample containing maize DNA with a pair of these primers which, when used in a nucleic acid amplification reaction with the genomic DNA of maize plants, produce a second amplicon containing the natural maize genomic DNA of the maize homologous to the genomic region of maize; and conducting a nucleic acid amplification reaction to produce a second amplicon. The methods further comprise detecting the second amplicon and comparing the first and second amplicons in the sample, where the presence of both amplicons indicates that the sample is heterozygous for the transgenic insert.

DNA detection kits can be prepared using the compositions described herein and methods well known in the field of DNA detection. The kits are effective for identifying the DNA of the subject corn object in the sample, and can be used in methods of crossing corn plants containing such DNA. The kits contain DNA sequences homologous or complementary to amplicons, such as those described herein, or DNA sequences homologous or complementary to the DNA contained in the transgenic genetic elements of the subject objects. These DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method. Kits may also contain reagents and materials necessary for the detection.

A “probe” is an isolated nucleic acid molecule coupled to a standard detectable label or reporter molecule (such as a radioactive isotope, ligand, chemiluminescent agent or enzyme). Such a probe is complementary to the target nucleic acid sequence, and in the case of the present invention, a genomic DNA sequence derived from one of these corn objects, or from a corn plant, or from a sample containing the DNA of this object. The probes of the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to the target DNA sequence and which can be used to detect the presence of such a target DNA sequence.

"Primers" are isolated / synthesized nucleic acids that are annealed with a complementary target DNA sequence to form a hybrid between the primer and the target DNA sequence, followed by extension along the target DNA strand by polymerase, for example, DNA polymerase. The primer pairs of the present invention are used to amplify a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

The length of the probes and primers is usually 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 , 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 , 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175 , 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 , 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 polynucleotides or more. Such probes and primers specifically hybridize to the target sequence under stringent hybridization conditions. Preferably, the sequences of the probes and primers of the present invention are completely identical to the target sequence, although traditional methods can be used to design probes that are different from the target sequence and retain the ability to hybridize with the target sequence.

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. Pairs of PCR primers can be obtained from a known sequence, for example, using computer programs designed for this purpose.

Primers and probes designed based on the flanking DNA and the insert sequences described herein can be used to confirm (and, if necessary, adjust) the sequences in question by traditional methods, for example, by re-cloning and sequencing of such sequences.

The nucleic acid probes and primers of the present invention hybridize under stringent conditions with the target DNA sequence. Any conventional method of nucleic acid hybridization or amplification can be used to detect the presence of a transgenic object in a DNA sample. Nucleic acid molecules or fragments thereof are capable of specific hybridization with other nucleic acid molecules under certain conditions. As used herein, it is indicated that two nucleic acid molecules are capable of specifically hybridizing with each other if said two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is considered “complementary” to another nucleic acid molecule if they are fully complementary. As used herein, it is believed that molecules are “fully complementary” if each nucleotide of one molecule is complementary to the nucleotide of another molecule. It is believed that two molecules have "minimal complementarity" if they can hybridize with each other with a stability sufficient to hybridize with each other, at least under standard "low stringency" conditions. Similarly, it is believed that molecules are "complementary" if they can hybridize with each other with a stability sufficient to hybridize with each other under standard "high stringency" conditions. Standard stiffness conditions are described in Sambrook et al., 1989. Thus, deviations from full complementarity are acceptable provided that such deviations do not in any way interfere with the ability of molecules to form double-stranded structures. For a nucleic acid molecule to serve as a primer or probe, it is only necessary that the degree of complementarity of the desired sequence is sufficient to form a stable double-stranded structure in a particular solvent and at specific salt concentrations.

As used herein, a substantially homologous sequence is a nucleic acid sequence that specifically hybridizes to a complement of the nucleic acid sequence with which it is compared under high stringency conditions. The term "stringent conditions" is functionally defined in relation to the hybridization of a nucleic acid probe with a target nucleic acid (i.e., with a specific nucleic acid sequence of interest) by the hybridization methods described in Sambrook et al., 1989, at 9.52-9.55. See also Sambrook et al., 1989 at 9.47-9.52 and 9.56-9.58. Thus, the nucleotide sequences of the invention can be used due to their ability to specifically form binary molecules with complementary regions of DNA fragments.

Depending on the application, various hybridization conditions can be used to achieve different degrees of specificity of the probe to the target sequence. For applications requiring high specificity, generally relatively stringent conditions are used to produce hybrids, for example, conditions with a relatively low salt concentration and / or high temperature, such as from about 0.02 M to about 0.15 M NaCl at a temperature of from about 50 ° C to about 70 ° C. Severe conditions, for example, may include at least washing the filter twice for hybridization with the washing buffer under high stringency conditions (0.2X SSC, 0.1% SDS, 65 ° C). Suitable stringency conditions that facilitate DNA hybridization, for example 6.0X sodium chloride / sodium citrate (SSC) at approximately 45 ° C followed by washing with 2.0X SSC at 50 ° C, are known to those skilled in the art. For example, the salt concentration in the washing step can vary from a concentration used under low hardness conditions of approximately 2.0X SSC at 50 ° C to a concentration used under high hardness conditions of approximately 0.2X SSC at 50 ° C. In addition, the temperature in the washing step can be increased from room temperature used under low hardness conditions of approximately 22 ° C to a temperature used in high hardness conditions of approximately 65 ° C. You can change both the temperature and salt concentration, or you can maintain the temperature at a constant level by changing the salt concentration, and vice versa. Such selective conditions do not allow mismatch, or allow only a slight mismatch, between the probe and the matrix circuit or target chain. Detection of a DNA sequence by hybridization is well known to those skilled in the art, and US Pat. Nos. 4,965,188 and 5,176,995 provide examples of methods for analyzing hybridization.

In a particularly preferred embodiment, the nucleic acid of the present invention specifically hybridizes to one or more primers (or amplicons or other sequences) provided by way of example or provided herein, including their complements and fragments, under high stringency conditions. In one aspect of the present invention, the nucleic acid molecule of the present invention has the nucleic acid sequence shown in SEQ ID NO: 2-7, or its complement and / or fragments.

In another aspect of the present invention, the sequence of the marker nucleic acid molecule of the present invention is 80-100% or 90-100% identical to the sequence of such a nucleic acid. In an additional aspect of the present invention, the sequence of the marker nucleic acid molecule of the present invention is 95-100% identical to such a sequence. Such sequences can be used as markers in plant crossing methods to identify offspring of genetic crosses. Hybridization of the probe with the target DNA molecule can be detected by various methods known to those skilled in the art, and these methods include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

Regarding amplification of a target nucleic acid sequence (for example, by PCR) using a specific pair of amplification primers, “stringent conditions” are conditions that allow a pair of primers to hybridize only with the target nucleic acid sequence to which the primer containing the corresponding a wild-type sequence (or its complement), with the preferred production of a unique amplification product, amplicon.

The term “specific for (target sequence)” means that the probe or primer hybridizes under stringent hybridization conditions only to the target sequence in the sample containing the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product of a nucleic acid amplification of a target nucleic acid sequence that is part of a template nucleic acid. For example, in order to determine whether a corn plant obtained by sexual intercourse contains the genomic DNA of a transgenic object derived from a corn plant of the present invention, DNA extracted from a tissue sample of a corn plant is subjected to nucleic acid amplification using a pair of primers that includes one primer originating from the flanking sequence in the genome of the plant adjacent to the insertion site of the inserted heterologous DNA, and a second primer derived from the inserted heterolog egg DNA to produce an amplicon, which is a diagnostic sign of the presence of transgenic DNA. The length and sequence of the amplicon are also diagnostic signs of the presence of an object. The length of such an amplicon can vary, making up the total length of the primer pairs plus one nucleotide pair and / or the total length of the primer pairs plus approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 , 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 , 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 , 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 750, 1000, 1250, 1500, 1750, 2000 or more pairs of nucleotides (plus or minus the elongation values given above). Alternatively, a pair of primers may be derived from the flanking sequence on both sides of the inserted DNA, so as to produce an amplicon including the entire inserted nucleotide sequence. A member of a primer pair derived from the genomic sequence of a plant may be located at a distance from the inserted DNA sequence. This distance can vary from one pair of nucleotides to about twenty thousand pairs of nucleotides. In particular, the use of the term “amplicon” excludes dimers of primers that can form in the thermal amplification reaction of DNA.

Amplification of nucleic acids can be carried out by various methods of amplification of nucleic acids known in the art, which include polymerase chain reaction (PCR). A number of amplification methods are known in the art and are described, including in US Pat. No. 4,683,195 and US Pat. No. 4,683,202. PCR amplification methods have been developed for amplification up to 22 kb. genomic DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the practice of the present invention. The sequence of the heterologous transgenic DNA insert or flanking genomic sequence derived from the subject of corn can be confirmed (and if necessary corrected) by amplification of such sequences of the specified object using primers derived from the sequences given in this document, followed by standard DNA sequencing PCR amplicon or cloned DNA.

The amplicon obtained by these methods can be detected in various ways. Agarose gel electrophoresis and ethidium bromide staining is a traditional well-known method for detecting DNA amplicons. Another such method is the analysis of the genetic code in which a DNA oligonucleotide is constructed that overlaps both the adjacent flanking genomic DNA sequence and the integrated DNA sequence. The oligonucleotide is immobilized in the wells of a microwell plate. After PCR of the desired region (using one primer in the inserted sequence and one primer in the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized with an immobilized oligonucleotide and it can serve as a matrix for the extension reaction to one base using DNA polymerase and labeled ddNTPs specific for the following alleged base. Reading data can be carried out by fluorescence analysis or ELISA analysis. Upon successful amplification, hybridization, and extension to one base, the signal indicates the presence of an insert / flanking sequence.

All patents, patent applications, provisional applications, and publications referenced or cited herein are hereby incorporated by reference in full to the extent that they are consistent with the idea of the present invention.

The abbreviations below apply unless otherwise indicated.

Aad-1 aryloxyalkanoate dioxygenase-1 bp nucleotide pair ° C degrees Celsius DNA Deoxyribonucleic acid Dig digoxygenin EDTA ethylenediaminetetraacetic acid tbp thousand pairs of nucleotides mcg micrograms μl microliter ml milliliter M molar mass OLP overlapping probe PCR polymerase chain reaction PTU plant transcription unit Sds sodium dodecyl sulfate Sop standard working method SSC buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 TBE buffer solution containing a mixture of tris, boric acid and EDTA, pH 8.3 V volt

EXAMPLES

Example 1. Object-specific analysis of Taqman

The Taqman object-specific analysis was designed to detect the presence of the DAS-40278-9 maize object and determine the type of plant zygosity in breeding populations. To conduct an object-specific analysis, specific Taqman primers and probes based on a DNA sequence located at the 5'-end of the junction with the transgene genome were developed. For specific detection, DAS-40278-9 amplified a 73 kb DNA fragment that encompasses the junction at the 5'end using two specific primers. Amplification of this PCR product was evaluated using a target-specific MGB probe synthesized by Applied Biosystems containing the FAM tag at the 5'-end. The specificity of this Taqman detection method for maize DAS-40278-9 AAD-1 was tested on 16 different maize AAD-1 and non-transgenic maize varieties using a maize-specific endogenous reference gene, the invertase gene.

Example 1.1. Isolation of gDNA

Tested gDNA samples from 16 different maize AAD-1 objects and non-transgenic maize varieties. gDNA was isolated using two methods, Qiagen kit and CTAB. Eight fresh pieces of corn leaf were used for the gDNA samples isolated using the Qiagen kit to isolate the gDNA according to the modified Qiagen DNeasy 96 Plant Kit technique. For gDNA samples isolated using the CTAB method, approximately 0.3 g of lyophilized leaf tissue was used according to the method described in Permingeat et al., 1998. The amount of gDNA was estimated using the Pico Green method according to the manufacturer's instructions (Molecular Probes, Eugene, OR). For the present study, gDNA samples were diluted with DNase-free water to obtain a concentration of 10 ng / μl.

Example 1.2 Taqman Analysis and Results

For Taqman analysis specific to the DAS-40278-9 object, specific Taqman primers and probes were designed. These reagents can be used under the conditions below to detect the corn object DAS-40278-9 AAD-1. Table 1 shows the sequences of the primer and probe, specially designed for the detection of the object DAS-40278-9.

Table 1
PCR primers and probes Target reaction Title Description Sequence from 5'-end to 3'-end Corn278-F Direct primer Seq ID NO: 2: 5 '- ATTCTGGCTTTGCTGTAAATCGT - 3' Corn278-R Reverse primer Seq ID NO: 3: 5 '- TTACAATCAACAGCACCGTACCTT - 3' Corn278-Probe Probe Seq ID NO: 4: 5 '- FAM-CTAACCTTCATTGTATTCC-MGB - 3' Invertase reference gene reaction Title Description Sequence from 5'-end to 3'-end IVF Direct primer Seq ID NO: 5: 5 '- TGGCGGACGACGACTTGT - 3' IVR Reverse primer Seq ID NO: 6: 5 '- AAAGTTTGGAGGCTGCCGT - 3' Iv-probe Probe Seq ID NO: 7: 5 '- HEX-CGAGCAGACCGCCGTGTACTTCTACC-BHQ2 - 3'

Multiple PCR conditions for amplification are as follows: 1X PCR buffer, 0.5-2.5 mM MgCl ₂ , 0.2 mM dNTP, 0.2 μM Corn-278-F primer, 0.2 μM Corn-278-R primer, 0.2 μM IV-F primer, 0.2 μM IV-R primer, 0.08 μM Corn-278-Probe probe, 0.08 μM IV-Probe probe, 40 U / ml HotStart Taq, 0.6-2 , 4 μg / ml DNA in the total reaction mixture with a volume of 25 μl. Different tested concentrations of MgCl ₂ and DNA. Applied concentrations of 0.5 mm, 1.0 mm, 1.8 mm and 2.5 mm MgCl ₂ . The mixture was amplified using the following conditions: i) 95 ° C for 15 min, ii) 95 ° C for 20 s, iii) 60 ° C for 60 s, iv) repeating steps ii-iii for 50 cycles, v ) retention at 4 ° C. Real-time PCR was performed on a Bio-rad iCycler ™ instrument. Data analysis was based on the measurement of the threshold cycle (CT), which is the PCR cycle number during which fluorescence reaches the set value. The CT value was calculated automatically using iCycler software.

The amplicon sequences obtained using the above primers are:

278F and 278R:

ttacaatcaacagcaccgtaccttgaagcggaatacaatgaaggttagctacgatttacagcaaagccagaat

(SEQ ID NO: 8)

IVF and IVR:

tggcggacgacgacttgtccgagcagaccgccgtgtacttctacctgctcaagggcacggacggcagcctccaaacttt

(SEQ ID NO: 9)

The Taqman method for detecting the AAD-1 maize object DAS-40278-9 was tested on 16 different maize AAD-1 objects and non-transgenic maize varieties using the maize-specific endogenous invertase gene as a reference gene. This analysis allowed the specific detection of the AAD-1 DAS-40278-9 maize object and did not yield any false positive results for control samples (i.e. 16 different AAD-1 maize objects and non-transgenic maize varieties). Object-specific primers and probes can be used to detect AAD-1 object DAS-40278-9 corn, as well as these conditions and reagents are applicable for zygosity analyzes.

Claims (15)

1. The method of determining the zygosity of a corn plant containing SEQ ID NO: 1 in the genome of corn,
where the specified genome of the corn contains a transgenic construct containing the aryloxyalkanoate dioxygenase gene (AAD-1),
where the specified transgenic design is flanked by 5′-flanking genomic DNA of the corn and 3′-flanking genomic DNA of the corn,
where the specified 5′-flanking genomic DNA of the corn consists of residues 1-1873 of the sequence SEQ ID NO: 1 and the specified 3′-flanking genomic DNA of the corn consists of residues 6690-8557 of the sequence SEQ ID NO: 1, and
where the specified transgenic construct consists of residues 1874-6689 of the sequence SEQ ID NO: 1,
where the specified method includes:
obtaining a sample of genomic DNA from the specified corn plant;
obtaining a contacted sample by contacting said DNA sample with
a) a first primer and a second primer, wherein said first primer comprises a segment of said transgenic construct, said second primer comprises a segment of said 5′-flanking genomic DNA of maize or a segment of said 3′-flanking genomic DNA of maize, and
where said first primer and said second primer produce an amplicon, which is then placed under quantitative PCR conditions,
b) a fluorescent probe that hybridizes with the specified amplicon;
placing said contacted sample under fluorescence based quantitative end-point PCR;
quantitative analysis of the specified fluorescent probe hybridized with the specified amplicon; and
determining the zygosity of said corn plant containing SEQ ID NO: 1. 2. The method of claim 1, wherein said amplicons are comprised of 50-100 residues. 3. The method of claim 1, wherein said second primer binds to residues 1673-1873 of the sequence SEQ ID NO: 1 or a sequence complementary thereto. 4. The method according to p. 1, where the second primer of the object is associated with residues 6690-6890 of the sequence SEQ ID NO: 1. 5. The method according to p. 1, where the specified method is used to analyze the introgression of the transgenic construct AAD-1 containing the sequence of SEQ ID NO: 1, for selection in another line of corn. 6. The method according to p. 5, where the specified other line of corn is devoid of the sequence of SEQ ID NO: 1. 7. The method according to p. 1, where the specified amplicon DAS-4 027 8-9 represents 73 base pairs. 8. The method of claim 1, wherein said probes are labeled with a fluorescent dye and quencher. 9. The method of claim 8, wherein said probe comprises FAM as said fluorescent dye at the 5′-end of said probe and an MGB quencher at the 3′-end of said probe. 10. The method according to p. 1, where the specified amplicon consists of the sequence of SEQ ID NO: 8. 11. The method according to p. 1, where the specified probe consists of SEQ ID NO: 4. 12. The method of claim 1, wherein said primers are selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 13. The method of claim 1, wherein the results of said zygosity of the corn plant are read directly on the tablet reader. 14. The method of claim 1, wherein said DNA sample is obtained from a corn plant in a field. 15. The kit for implementing the method according to claim 1, wherein said kit includes said primers consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and said probe, consisting of SEQ ID NO: 4.

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