Endpoint TaqMan methods for determining zygosity of cotton comprising Cry1Ac event 3006-210-23
View Patent ↗A method for zygosity analysis of the cotton Cry1Ac event 3006-210-23 is provided. The method provides 3006-210-23 event-specific and cotton-genome-specific primers and TaqMan probe combinations for use in an endpoint biplex TaqMan PCR assay capable of determining event zygosity and for assisting in event introgression and breeding.
1. A method for determining event zygosity of a cotton plant comprising a 3006-210-23 event, said 3006-210-23 event comprising a transgene construct comprising a cry1Ac gene, said method comprising:
obtaining a sample of genomic DNA from said cotton plant,
contacting said sample with
a. a first flanking primer consisting of SEQ ID NO:2;
b. a second flanking primer consisting of SEQ ID NO:4;
c. a transgene primer consisting of SEQ ID NO:3;
wherein said first flanking primer and said second flanking primer form a wild-type amplicon when subjected to PCR conditions,
wherein said transgene primer forms a transgene amplicon with said first flanking primer or said second flanking primer when subjected to PCR conditions,
further contacting said sample with
d. a florescent event probe that hybridizes with said transgene amplicon
e. a florescent wild-type probe that hybridizes with said wild-type amplicon
subjecting said sample to fluorescence-based endpoint TaqMan PCR conditions, quantitating said florescent event probe that hybridized to said event amplicon,
quantitating said florescent wild-type probe that hybridized to said wild-type amplicon,
comparing amounts of hybridized florescent event probe to hybridized florescent wild-type probe; and
determining zygosity of said cotton tissue by comparing florescence ratios of hybridized fluorescent event probe and hybridized fluorescent wild-type probe.
2. The method of claim 1 , wherein said plant comprises a first subgenome and a second subgenome, said transgene amplicon being formed from said first subgenome, and said wild-type amplicon being formed from said second subgenome.
3. The method of claim 1 wherein results of said method are read directly in a plate reader.
4. The method of claim 1 wherein said probes are labeled with a fluorescent dye and quencher.
5. The method of claim 1 wherein said transgene probe comprises TEXAS RED™ as said fluorescent dye at the 5′ end of said transgene probe and BLACK HOLE QUENCHER (BHQ™) as said quencher on the 3′ end of said transgene probe.
6. The method of claim 1 wherein said wild-type probe is labeled with a fluorescent dye (FAM) at the 5′ end of said wild-type probe and a BLACK HOLE QUENCHER (BHQ™) as said at the 3′ end of said wild-type probe.
7. The method of claim 1 wherein said wild-type probe comprises SEQ ID NO:6.
8. The method of claim 1 wherein said transgene probe comprises SEQ ID NO:5.
9. The method of claim 1 wherein said transgene amplicon comprises SEQ ID NO:8.
10. The method of claim 1 wherein said wild-type amplicon comprises SEQ ID NO:9.