Elite Event A2407-12 and Methods and Kits for Identifying Such Event in Biological Samples

• US 20080320616A1
• Filed: 04/04/2006
• Published: 12/25/2008
• Est. Priority Date: 04/08/2005
• Status: Active Grant

• Current Assignee: BASF Agricultural Solutions Seed US LLC (BASF SE)
• Original Assignee: Bayer Bioscience NV (Bayer AG)
• Inventors: De Beuckeleer, Marc
• Granted Patent: US 8,012,689 B2
• Time in Patent Office: Days
• US Class Current: 800/312
• CPC Class Codes: C12N 15/8277 Phosphinotricin
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

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

Claims

1. A method for identifying elite event A2704-12 in biological samples, comprising detecting 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 bp 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 comprising 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 comprising 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 comprising 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 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 said primer recognizing the 3′

flanking region of A2704-12 consists 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 primer recognizing the 5′

flanking region comprises the sequence of SEQ ID No. 4 and said primer recognizing a sequence within the foreign DNA comprises SEQ ID No. 8.

6. The method of claim 5, which method comprises amplifying a fragment of about 185 bp using the A2704-12 identification protocol.

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.

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 comprising 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 comprising 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 comprising 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 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 said primer recognizing the 3′

flanking region of A2704-12 consists 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, comprising 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 comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 and said 3′

flanking region comprising 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.

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, comprising detecting 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, comprising detecting 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.