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

• US 20120117693A1
• Filed: 08/04/2011
• Published: 05/10/2012
• 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 8,952,142 B2
• Time in Patent Office: Days
• US Class Current: 800/300
• 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 of elite event A5547-127 in biological samples.

Claims

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

or 3′

flanking region of A5547-127.

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

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 having 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 having 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 having 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 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 311 or said primer recognizing the 3′

flanking region of A5547-127 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 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, having 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 having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311 and said 3′

flanking region having 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.

16. (canceled)

17. (canceled)

18. (canceled)

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, the sequence of being 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. (canceled)

27. (canceled)

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

29. A DNA

(a) comprising a sequence of between 50 and 500 bp with at least 80% sequence identity to a DNA comprising the 5′

flanking region of the A5547-127 event and the foreign DNA contiguous therewith, said 5′

flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311, and said foreign DNA contiguous therewith comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 312 to nucleotide 810;

(b) comprising a sequence of between 50 and 500 bp with at least 80% sequence identity to a DNA comprising the 3′

flanking region of the A5547-127 event and the foreign DNA contiguous therewith, said 3′

flanking region comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, and said the foreign DNA contiguous therewith comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509;

or

(c) specifically identifying soybean elite event A5547-127, said DNA comprises the nucleotide sequence of a discriminating amplicon obtainable from a nucleic acid sample comprising said elite event using a polymerase chain reaction with a primer specifically recognizing a sequence within the 5′

or 3′

flanking region of the elite event and a primer specifically recognizing the DNA contiguous therewith, wherein said 5′

flanking region comprises the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 311, the foreign DNA contiguous therewith comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 312 to nucleotide 810, and wherein said 3′

flanking region of A5547-127 comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, and the foreign DNA contiguous therewith comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509.