Elite Event A5547-127 and Methods and Kits For Identifying Such Event in Biological Samples

• US 20080196127A1
• Filed: 04/04/2006
• Published: 08/14/2008
• Est. Priority Date: 04/11/2005
• 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 8,017,756 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 A5547-127 in biological samples.

Claims

1. A method for identifying elite event A5547-127 in biological samples, comprising detecting a A5547-127 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of A5547-127.

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 A5547-127, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311 or the 3′

flanking region of A5547-127, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

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 311 or said primer recognizing the 3′

flanking region of A5547-127 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 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

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 311 or said primer recognizing the 3′

flanking region of A5547-127 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 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

5. The method of claim 4, wherein said primer recognizing a sequence within the foreign DNA comprises the sequence of SEQ ID No. 13 and wherein said primer recognizing the 5′

flanking region comprises SEQ ID No. 15.

6. The method of claim 5, which method comprises amplifying a fragment of about 151 bp using the A5547-127 identification protocol.

16. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with a specific probe for A5547-127.

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 A5547-127 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 260 to 360 or SEQ ID No. 2 from nucleotide 460 to 560, or the complement of said sequences.

7. A kit for identifying elite event A5547-127 in biological samples, said kit comprising one primer recognizing the 5′

flanking region of A5547-127, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311 or one primer recognizing the 3′

flanking region of A5547-127, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

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 311 or said primer recognizing the 3′

flanking region of A5547-127 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 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

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 311 or said primer recognizing the 3′

flanking region of A5547-127 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 510 to nucleotide 1880, 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 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

10. The kit of claim 7, comprising a primer consisting of the sequence of SEQ ID No. 13 and a primer consisting of the sequence of SEQ ID No. 15.

11. A primer for use in a A5547-127 PCR identification protocol, comprising a sequence which, under optimized PCR conditions specifically recognizes a sequence within the 5′

or 3′

flanking region of A5547-127, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311 and said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880.

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 311 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 510 to nucleotide 1880.

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 311 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 510 to nucleotide 1880.

14. A primer comprising at its extreme 3′

end the sequence of SEQ ID No. 13.

15. A primer comprising at its extreme 3′

end the sequence of SEQ ID No. 15.

19. A kit for identifying elite event A5547-127 in biological samples, said kit comprising a specific probe, capable of hybridizing specifically to a specific region of A5547-127.

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 A5547-127 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 260 to 360 or SEQ ID No. 2 from nucleotide 460 to 560, or the complement of said sequences.

22. A specific probe for the identification of elite event A5547-127 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 A5547-127 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 260 to 360 or SEQ ID No. 2 from nucleotide 460 to 560, or the complement of said sequences.

25. A specific probe for the identification of elite event A5547-127 in biological samples, wherein said specific probe is essentially similar to SEQ ID No. 1 from nucleotide 260 to 360 or SEQ ID No. 2 from nucleotide 460 to 560, or the complement of said sequences.

26. A method for confirming seed purity, comprising detecting a A5547-127 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of A5547-127, in seed samples.

27. A method for screening seeds for the presence of A5547-127, comprising detecting a A5547-127 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of A5547-127, in samples of seed lots.

28. A soybean plant, or cells, parts, seed or progeny thereof, comprising elite event A5547-127 in its genome.