Materials and Methods for Detecting the Aryloxyalkanoate Dioxygenase Gene (AAD-12) Containing Event pDAB4472-1606 in Plants
AssigneeDow AgroSciences LLC
InventorsCHANDRA-SHEKARA A. CHANNABASAVARADHYA, ANDREW GREENWALD
Life of this patent
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Abstract
This application provides materials and methods for the detection of the aad-12 gene and event pDAB4472-1606 in biological samples derived from plants.
Claims21
An isolated primer or probe comprising SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, SEQ ID NO:
- 4 or SEQ ID NO:
- 5.
A kit comprising a container and a composition comprising a combination of nucleic acid sequences selected from:
- a) SEQ ID NO:
- 1, SEQ ID NO:
- 2 and SEQ ID NO:
- 3;
- b) SEQ ID NO:
- 1, SEQ ID NO:
- 4 and SEQ ID NO:
- 5;
- c) SEQ ID NO:
- 1 and SEQ ID NO:
- 2;
- d) SEQ ID NO:
- 4 and SEQ ID NO:
- 5;
- e) SEQ ID NO:
- 1 and SEQ ID NO:
- 3;
- f) SEQ ID NO:
- 2 and SEQ ID NO:
- 3;
- g) SEQ ID NO:
- 1 and SEQ ID NO:
- 4;
- h) SEQ ID NO:
- 1 and SEQ ID NO:
- 5;
- i) SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- j) SEQ ID NO:
- 2 and SEQ ID NO:
- 5;
- k) SEQ ID NO:
- 3 and SEQ ID NO:
- 4;
- or
- l) SEQ ID NO:
- 3 and SEQ ID NO:
- 5.
A method of determining zygosity of DNA of a soybean plant genome comprises (a) contacting a biological sample with a primer pair of SEQ ID NOs:
- 1 and 2, that when used in a nucleic acid amplification reaction with genomic DNA from a biological sample containing genomic DNA from a biological sample from a soybean plant containing event pDAB4472-1606, produces a first amplicon that identifies the presence of the aad12 gene within the genomic DNA of the biological sample;
- (b) performing a nucleic acid amplification reaction;
- (c) detecting a first amplicon produced;
- (d) contacting the same sample with a second primer pair of SEQ ID NOs:
- 1 and 4, that when used in a nucleic acid amplification reaction with a biological sample containing genomic DNA from soybean plants produces an amplicon from the combination of primers that identifies wild-type soybean genomic DNA homologous to the soybean genomic region of a transgene insertion identified as event pDAB4472-1606;
- (e) performing a nucleic acid amplification reaction, and (f) detecting a second amplicon produced;
- wherein:
- (i) detection of both amplicons indicates that the soybean sample is heterozygous for event pDAB4472-1606;
- (ii) detection of only said first amplicon (identifying the presence of the aad12 gene) indicates the biological sample is homozygous for event pDAB4472-1606;
- and (iii) detection of only said second amplicon is indicative of the absence of event pDAB4472-1606 within the biological sample.
A method of detecting the presence of DNA corresponding to the event pDAB4472-1606 in a biological sample comprising:
- (a) contacting the biological sample with a primer pair of SEQ ID NOs:
- 1 and 2 that, when used in a nucleic acid amplification reaction with genomic DNA obtained from one or more soybean plant(s) containing event pDAB4472-1606, produces an amplicon that is identifies the plant as containing event pDAB4472-1606;
- (b) performing a nucleic acid amplification reaction, thereby producing the amplicon;
- and
- (c) detecting the amplicon with a probe that is, optionally, labeled, wherein detection of said amplicon is indicative of presence of the DNA corresponding to event pDAB4472-1606.
A method of detecting the presence of a DNA corresponding to event pDAB4472-1606 in a biological sample comprising:
- (a) contacting the biological sample with a probe that hybridizes under stringent hybridization conditions with genomic DNA from a soybean plant containing event pDAB4472-1606 and does not hybridize under the stringent hybridization conditions with a control soybean plant, said probe being optionally labeled and comprising SEQ ID NOs:
- 2 or 3;
- (b) subjecting the biological sample and probe to stringent hybridization conditions;
- and
- (c) detecting hybridization of the probe to the DNA containing event pDAB4472-1606, wherein detection of such hybridization in indicative of presence of the DNA corresponding to event pDAB4472-1606 within the genome of the plant.
A method of amplifying nucleic acids in a biological sample to identify the presence of event pDAB4472-1606 within the genome of a soybean plant comprising:
- (a) contacting a biological sample with a primer pair of SEQ ID NOs:
- 1 and 2, that when used in a nucleic acid amplification reaction with genomic DNA from a biological sample containing genomic DNA from a biological sample from a soybean plant containing event pDAB4472-1606, produces an that identifies the presence of the aad-12 gene within the genomic DNA of the biological sample;
- (b) performing a nucleic acid amplification reaction;
- and (c) detecting an amplicon produced by said amplification reaction, wherein detection of said amplicon indicates the biological sample contains the aad-12 gene.
A composition comprising a buffer and a combination of nucleic acid sequences selected from:
- a) SEQ ID NO:
- 1, SEQ ID NO:
- 2 and SEQ ID NO:
- 3;
- b) SEQ ID NO:
- 1, SEQ ID NO:
- 4 and SEQ ID NO:
- 5;
- c) SEQ ID NO:
- 1 and SEQ ID NO:
- 2;
- d) SEQ ID NO:
- 4 and SEQ ID NO:
- 5;
- e) SEQ ID NO:
- 1 and SEQ ID NO:
- 3;
- f) SEQ ID NO:
- 2 and SEQ ID NO:
- 3;
- g) SEQ ID NO:
- 1 and SEQ ID NO:
- 4;
- h) SEQ ID NO:
- 1 and SEQ ID NO:
- 5;
- i) SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- j) SEQ ID NO:
- 2 and SEQ ID NO:
- 5;
- k) SEQ ID NO:
- 3 and SEQ ID NO:
- 4;
- or
- l) SEQ ID NO:
- 3 and SEQ ID NO:
- 5.
Dependencies are inferred from the claim text.