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US 2013/0095486 A1Published application
Materials and Methods for Detecting the Aryloxyalkanoate Dioxygenase Gene (AAD-12) in Plants
AssigneeDow AgroSciences LLC
InventorsCHANDRA-SHEKARA A. CHANNABASAVARADHYA, ANDREW GREENWALD
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Abstract
This application provides materials and methods for the detection of aad-12 gene events in biological samples derived from recombinant plants and a materials and methods for the detection of contaminating events in samples derived from recombinant plants.
Claims21
An isolated primer or probe comprising SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, SEQ ID NO:
- 4, SEQ ID NO:
- 5 or SEQ ID NO:
- 6.
3 more elements
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:
- 4, SEQ ID NO:
- 5 and SEQ ID NO:
- 6;
- 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:
- 4 and SEQ ID NO:
- 6;
- h) SEQ ID NO:
- 1 and SEQ ID NO:
- 4;
- i) SEQ ID NO:
- 1 and SEQ ID NO:
- 5;
- j) SEQ ID NO:
- 1 and SEQ ID NO:
- 6;
- k) SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- l) SEQ ID NO:
- 2 and SEQ ID NO:
- 5;
- m) SEQ ID NO:
- 2 and SEQ ID NO:
- 6;
- n) SEQ ID NO:
- 3 and SEQ ID NO:
- 4;
- o) SEQ ID NO:
- 3 and SEQ ID NO:
- 5;
- or
- p) SEQ ID NO:
- 3 and SEQ ID NO:
- 6.
48 more elements
A method of determining zygosity of DNA of a soybean plant genome 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 a first amplicon that identifies the presence of the aad-12 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:
- 4 and 5, 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 aad-12 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.
7 more elements
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 set 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 wherein detection of said amplicon is indicative of presence of the DNA corresponding to event pDAB4472-1606.
1 more element
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;
- (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.
1 more element
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.
1 more element
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:
- 4, SEQ ID NO:
- 5 and SEQ ID NO:
- 6;
- 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:
- 4 and SEQ ID NO:
- 6;
- h) SEQ ID NO:
- 1 and SEQ ID NO:
- 4;
- i) SEQ ID NO:
- 1 and SEQ ID NO:
- 5;
- j) SEQ ID NO:
- 1 and SEQ ID NO:
- 6;
- k) SEQ ID NO:
- 2 and SEQ ID NO:
- 4;
- l) SEQ ID NO:
- 2 and SEQ ID NO:
- 5;
- m) SEQ ID NO:
- 2 and SEQ ID NO:
- 6;
- n) SEQ ID NO:
- 3 and SEQ ID NO:
- 4;
- o) SEQ ID NO:
- 3 and SEQ ID NO:
- 5;
- or
- p) SEQ ID NO:
- 3 and SEQ ID NO:
- 6.
48 more elements
A method for determining contaminating events in a recombinant plant comprising:
- (a) contacting a recombinant plant DNA sample consisting of a DNA fragment containing a transgene contiguously linked to a genomic DNA with a first primer pair that hybridize to said DNA fragment containing a transgene contiguously linked to a genomic DNA (b) performing a nucleic acid amplification reaction;
- (c) producing a first amplicon consisting of said DNA fragment containing a transgene contiguously linked to a genomic DNA;
- (d) detecting the first amplicon;
- (e) contacting said recombinant plant DNA sample consisting of a DNA fragment containing a native homozygous gene with a second primer pair that hybridize to said DNA fragment containing a native homozygous gene;
- (f) performing a nucleic acid amplification reaction;
- (g) producing a second amplicon consisting of said native homozygous gene;
- (h) detecting the second amplicon;
- (i) determining that signal strengths from said first and second amplicons are approximately equal, wherein equal signal strengths of the first and second amplicon indicate the absence of contaminating events;
- (j) contacting said recombinant plant DNA sample consisting of a DNA fragment containing a transgene with a third primer pair that hybridize to said DNA fragment containing a transgene;
- (k) performing a nucleic acid amplification reaction;
- (1) producing a third amplicon consisting of said transgene;
- (m) detecting the third amplicon, and;
- (n) determining that signal strengths of first and third amplicons are approximately equal, wherein equal signal strengths of the first and third amplicon indicate the absence of contaminating events.
10 more elements
Dependencies are inferred from the claim text.