Elite event a2704-12 and methods and kits for identifying such event in biological samples

Abstract

Tools are provided which allow rapid and unequivocal identification elite event A2704-12 in biological samples.

1 . A method for identifying elite event A2704-12 in biological samples, which method comprises detection of a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12. 
     
     
         2 . The method of  claim 1 , said method comprising amplifying a DNA fragment of between 100 and 500 by from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ flanking region of A2704-12, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or the 3′ flanking region of A2704-12, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, the other primer of said primers recognizing a sequence within the foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         3 . The method of  claim 2 , wherein said primer recognizing the 5′ flanking region consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         4 . The method of  claim 2 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         5 . The method of  claim 4 , wherein said primers comprise the sequence of SEQ ID No. 4 and SEQ ID No. 8, respectively. 
     
     
         6 . The method of  claim 5 , which method comprises amplifying a fragment of about 185 by using the A2704-12 identification protocol, 
     
     
         7 . A kit for identifying elite event A2704-12 in biological samples, said kit comprising one primer recognizing the 5′ flanking region of A2704-12, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or one primer recognizing the 3′ flanking region of A2704-12, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and one primer recognizing a sequence within the foreign DNA, said foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         8 . The kit of  claim 7 , wherein said primer recognizing the 5′ flanking region consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         9 . The kit of  claim 7 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         10 . The kit of  claim 7 , comprising a primer consisting of the sequence of SEQ ID No. 4 and a primer consisting of the sequence of SEQ ID No. 8. 
     
     
         11 . A primer for use in a A2704-12 PCR identification protocol, having a sequence which, under optimized PCR conditions specifically recognizes a sequence within the 5′ or 3′ flanking region of A2704-12, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 and said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         12 . The primer of  claim 11 , wherein said primer consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         13 . The primer of  claim 11 , wherein said primer comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000. 
     
     
         14 . A primer comprising at its extreme 3′ end the sequence of SEQ ID No. 4. 
     
     
         15 . A primer comprising at its extreme 3′ end the sequence of SEQ ID No. 8. 
     
     
         16 . The method of  claim 1 , which method comprises hybridizing a nucleic acid of biological samples with a specific probe for A2704-12. 
     
     
         17 . The method of  claim 16 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith. 
     
     
         18 . The method of  claim 17 , wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences. 
     
     
         19 . A kit for identifying elite event A2704-12 in biological samples, said kit comprising a specific probe, capable of hybridizing specifically to a specific region of A2704-12. 
     
     
         20 . The kit of  claim 19 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith. 
     
     
         21 . The kit of  claim 20 , wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences. 
     
     
         22 . A specific probe for the identification of elite event A2704-12 in biological samples. 
     
     
         23 . The probe of  claim 22 , which has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith, or the complement thereof. 
     
     
         24 . The probe of  claim 23  which has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences 
     
     
         25 . A specific probe for the identification of elite event A2704-12 in biological samples, the sequence of being essentially similar to SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences. 
     
     
         26 . A method for confirming seed purity, which method comprises detection of a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12, in seed samples. 
     
     
         27 . A method for screening seeds for the presence of A2704-12, which method comprises detection of a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12, in samples of seed lots. 
     
     
         28 . A soybean plant, or cells, parts, seed or progeny thereof, comprising elite event A2704-12 in its genome.