	US 12,252,721 B2
	Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof
Yunxing C. Cui, Carmel, IN (US); Thomas Hoffman, Zionsville, IN (US); Ning Zhou, Zionsville, IN (US); Stephen N. Novak, Westfield, IN (US); Julissa Colon, Cidra, PR (US); Dawn M. Parkhurst, Avon, IN (US); Sandra G. Toledo, West Lafayette, IN (US); Terry R. Wright, Westfield, IN (US); Sean M. Russell, Carmel, IN (US); Bruce Held, Ames, IA (US); and Vaithilingam Sekar, Ames, IA (US)
Assigned to CORTEVA AGRISCIENCE LLC, Indianapolis, IN (US); and M.S. TECHNOLOGIES, L.L.C., West Point, IA (US)
Filed by CORTEVA AGRISCIENCE LLC, Indianapolis, IN (US); and M.S. TECHNOLOGIES, L.L.C., West Point, IA (US)
Filed on Nov. 8, 2023, as Appl. No. 18/504,940.
Application 18/504,940 is a division of application No. 17/897,988, filed on Aug. 29, 2022, granted, now 11,819,022.
Application 17/897,988 is a division of application No. 17/224,694, filed on Apr. 7, 2021, granted, now 11,425,906, issued on Aug. 30, 2022.
Application 17/224,694 is a division of application No. 16/434,995, filed on Jun. 7, 2019, granted, now 10,973,229, issued on Apr. 13, 2021.
Application 16/434,995 is a division of application No. 15/399,674, filed on Jan. 5, 2017, granted, now 10,400,250, issued on Sep. 3, 2019.
Application 15/399,674 is a continuation of application No. 13/991,246, granted, now 9,540,655, issued on Jan. 10, 2017, previously published as PCT/US2011/063129, filed on Dec. 2, 2011.
Claims priority of provisional application 61/521,798, filed on Aug. 10, 2011.
Claims priority of provisional application 61/511,664, filed on Jul. 26, 2011.
Claims priority of provisional application 61/471,845, filed on Apr. 5, 2011.
Claims priority of provisional application 61/419,706, filed on Dec. 3, 2010.
Prior Publication US 2024/0147996 A1, May 9, 2024
Int. Cl. C12N 15/82 (2006.01); C12N 9/00 (2006.01); C12N 9/10 (2006.01); C12Q 1/6895 (2018.01); A01N 39/04 (2006.01)

CPC C12N 9/93 (2013.01) [C12N 9/1092 (2013.01); C12N 15/8241 (2013.01); C12N 15/8274 (2013.01); C12N 15/8275 (2013.01); C12Q 1/6895 (2013.01); C12Y 205/01019 (2013.01); C12Y 604/01002 (2013.01); A01N 39/04 (2013.01); C12Q 2600/13 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/16 (2013.01)]

16 Claims

1. A method for determining event zygosity of a soybean plant comprising soybean Event pDAB8264.44.06.1, as present in seed deposited under ATCC Accession number PTA-11336, said Event pDAB8264.44.06.1 comprising a transgene construct, said transgene construct being flanked by a 5′ flanking soybean genomic DNA and a 3′ flanking soybean genomic DNA, said method comprising:

obtaining a DNA sample comprising genomic DNA from said soybean plant;

producing a contacted sample by contacting said DNA sample with

a. a first event primer and a second event primer, wherein said first event primer specifically binds said transgene construct, said second event primer specifically binds said 5′ flanking soybean genomic DNA or said 3′ flanking soybean genomic DNA, and wherein said first event primer and said second event primer produce an event amplicon when subjected to PCR conditions;

b. a reference forward primer and a reference reverse primer that produce a reference amplicon from an endogenous soybean reference gene when subjected to PCR conditions;

c. a florescent event probe that is labeled with a fluorescent dye and a quencher and hybridizes with said event amplicon;

d. a florescent reference probe that is labeled with a fluorescent dye and a quencher and hybridizes with said reference amplicon;

subjecting said contacted sample to fluorescence-based endpoint PCR conditions in the presence of a thermostable polymerase, dNTPs, said fluorescent event probe and said fluorescent reference probe;

quantitating said florescent event probe that hybridized to said event amplicon;

quantitating said florescent reference probe that hybridized to said reference amplicon;

comparing amounts of hybridized florescent event probe to amounts of hybridized florescent reference probe; and

determining zygosity of said Event pDAB8264.44.06.1 by comparing florescence ratios of said hybridized fluorescent event probe and said hybridized fluorescent reference probe.