Elite event ee-gh7 and methods and kits for identifying such event in biological samples
AssigneeBayer CropScience AG
InventorsStefan JANSENS, Rozemarijn DREESEN, Wendy AARTSEN, Jonas VANHAELEN, Hal MOSER, Ginger LIGHT
Life of this patent
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Abstract
The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific herbicide tolerance transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.
Claims68
A nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 1 from nucleotide 1207 to nucleotide 1228 or SEQ ID No. 1 from nucleotide 8022 to 8043, or the complement of said sequences.
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Cotton genomic DNA obtainable from the cotton plant, cell part, tissue, seed or progeny thereof of claim 9, said cotton genomic DNA comprising elite event EE-GH7.
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A cotton plant, cell, part, tissue, seed or progeny thereof, comprising elite event EE-GH7 in its genome.
Seed comprising elite event EE-GH7 deposited at the ATCC under deposit number PTO-122856 or derivatives therefrom.
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A method for identifying elite event EE-GH7 in biological samples, which method comprises detection of an EE-GH7 specific region with a specific primer pair or probe which specifically recognizes the 5′ or 3′ flanking region of the foreign DNA comprising herbicide tolerance genes in EE-GH7, and part of the foreign DNA contiguous with said 5′ or 3′ flanking region.
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A kit comprising a first and a second primer, said first primer recognizing the 5′ flanking region of the foreign DNA comprising herbicide tolerance genes in EE-GH7, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1217 or said first primer recognizing the 3′ flanking region of the foreign DNA comprising herbicide tolerance genes in EE-GH7, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 8033 to nucleotide 9328, and said second primer recognizing a sequence within the foreign DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1218 to nucleotide 8032 or the complement thereof.
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A kit for identifying the elite event EE-GH7 in biological samples, said kit comprising a specific probe which has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GG7 and the sequence of the foreign DNA contiguous therewith.
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A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH7, said method comprising amplifying DNA fragments of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plan DNA, such as a primer comprising the nucleotide sequence of SEQ ID No.11 and a primer comprising the nucleotide sequence of SEQ ID No. 12, the third of said primers recognizing a sequence within the foreign DNA, such as the nucleotide sequence of SEQ ID No. 13.
A method of detecting the presence of elite event EE-GH7 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising:
- a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1218 to nucleotide 1235 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 8015 to 8032 or its complement;
- b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1200 to nucleotide 1217 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 8033 to nucleotide 8050 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;
- c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;
- d) recycling of the target nucleic acid sequence by repeating steps (a) to (c);
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- e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.
Dependencies are inferred from the claim text.