Cotton event pv-ghbk04(531) and compositions and methods for detection thereof

Abstract

The present invention provides assays for detecting the presence of the 531 cotton event nucleic acid sequences in a biological sample based on the DNA sequence of the recombinant construct inserted into the cotton genome and of genomic sequences flanking the insertion site in a cotton genome.

1 . An isolated polynucleotide comprising the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, and complements thereof.  
     
     
         2 . A first and a second polynucleotide primer which function together in the presence of template cotton plant event 531 DNA in a sample to produce an amplicon diagnostic for the cotton plant event 531, said first and second polynucleotide primers being selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and complements thereof.  
     
     
         3 . The first and second polynucleotide primers of  claim 2  wherein said first polynucleotide primer comprises SEQ ID NO: 19 and said second polynucleotide primer comprises SEQ ID NO: 20, and wherein said amplicon comprises SEQ ID NO: 2.  
     
     
         4 . The first and second polynucleotide primers of  claim 2  wherein said first polynucleotide primer is selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 17 and said second polynucleotide primer is selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 18, and wherein said amplicon comprises SEQ ID NO: 1.  
     
     
         5 . The first and second polynucleotide primers of  claim 2  wherein said first polynucleotide primer is selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 27 and said second polynucleotide primer is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 28, and wherein said amplicon comprises a polynucleotide sequence selected from SEQ ID NO: 32 and SEQ ID NO: 4.  
     
     
         6 . The first and second polynucleotide primers of  claim 2  wherein said first polynucleotide primer is the complement of a cotton plant genome DNA flanking the point of insertion of a heterologous DNA sequence inserted into the cotton plant genome, and said second polynucleotide primer is the complement of a part of the heterologous DNA sequence inserted into the cotton plant genome, and wherein said amplicon is diagnostic for said event 531.  
     
     
         7 . A method of detecting the presence of cotton plant event 531 DNA in a sample comprising the steps of: 
 a) contacting said sample with a first polynucleotide primer and a second polynucleotide primer which function together in the presence of template cotton plant event 531 DNA in a sample to produce an amplicon diagnostic for the cotton plant event 531;    b) performing a nucleic acid amplification reaction, thereby producing said amplicon; and    c) detecting said amplicon.    
     
     
         8 . The method of  claim 7  wherein said first and second polynucleotide primers are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and complements thereof.  
     
     
         9 . The method of  claim 7  wherein said amplicon comprises a nucleotide sequence comprising at least 11 consecutive nucleotides selected fro mteh group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, and complements thereof.  
     
     
         10 . An isolated polynucleotide molecule comprising the amplicon produced by the method of  claim 7 .  
     
     
         11 . A nucleic acid detection kit for use in identifying the presence of cotton plant event 531 nucleic acids in a sample comprising: 
 a) a probe which is or is complementary to a part of the heterologous DNA sequence present in the genome of cotton plant event 531, said probe comprising at least 11 or more consecutive nucleotides, said consecutive nucleotides being selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, and complements thereof,    b) reagents necessary for detecting the binding of said probe to the heterologous DNA sequence inserted into the genome of cotton plant event 531; and    c) instructions for use; packaged together in said kit.    
     
     
         12 . A method of detecting cotton plant event 531 DNA in a sample comprising 
 a) contacting the sample with a polynucleotide probe that binds under stringent conditions with said DNA;    b) subjecting the sample and probe to said stringent conditions; and    c) detecting the binding of the probe to said DNA.    
     
     
         13 . An isolated nucleic acid comprising a di-nucleotide linking a heterologous DNA molecule to the cotton plant genome in cotton plant event 531 comprising a sequence of 11 consecutive nucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, and complements thereof.  
     
     
         14 . A method of detecting the presence of a DNA molecule corresponding to a DNA sequence in cotton plant event 531 or homozygous or heterozygous progeny thereof in a sample, the method comprising: 
 a) contacting the sample with a probe that does not bind under stringent conditions with DNA from a cotton plant other than said event;    b) subjecting the probe and sample to stringent conditions; and    c) detecting the binding of the probe to said DNA molecule.    
     
     
         15 . A method of determining the zygosity of a cotton plant genome comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 32, and complements thereof, said method comprising the steps of: 
 a) contacting a sample comprising DNA obtained from said cotton plant with a first and a second nucleotide primer that, when used in a nucleic acid amplification reaction with a first template comprising cotton plant event 531 DNA, produces a first amplicon that is diagnostic for cotton plant event 531;    b) performing a nucleic acid amplification reaction with said first and second nucleotide primers and said first template, thereby producing said first amplicon;    c) detecting said first amplicon;    d) contacting said sample with a first and a third nucleotide primer that, when used in a nucleic acid amplification reaction with a second template comprising cotton plant DNA other than event 531 DNA, produces a second amplicon that is diagnostic for other than cotton plant event 531;    e) performing a nucleic acid amplification reaction with said first and third nucleotide primers and said second template, thereby producing said second amplicon; and    f) detecting said second amplicon,    wherein detection of said second amplicon is diagnostic for the zygosity of said cotton plant genome.