Elite event A5547-127 and methods and kits for identifying such event in biological samples

• US 8,952,142 B2
• Filed: 08/04/2011
• Issued: 02/10/2015
• Est. Priority Date: 04/11/2005
• Status: Active Grant

• Current Assignee: BASF Agricultural Solutions Seed US LLC (BASF SE)
• Original Assignee: Bayer Cropscience NV (Bayer AG)
• Inventors: de Beuckeleer, Marc
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US13/198,639
• Publication Number:
  US 20120117693A1
• Time in Patent Office: 1,286 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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 kit comprising a primer pair, one primer of said pair recognizing the 5′

flanking region of A5547-127, and the other primer of said pair recognizing the foreign DNA contiguous with said 5′

flanking region, or one primer of said pair recognizing the 3′

flanking region of A5547-127 and the other primer of said pair recognizing the foreign DNA contiguous with said 3′

flanking region,

wherein said 5′

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

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, wherein said foreign DNA contiguous with said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509.

2. The kit of claim 1, wherein said first 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, and said second 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 said first 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 second primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 2 from 1 to nucleotide 509.

3. The kit of claim 1, wherein said first 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, and second primer recognizing a sequence within the foreign DNA 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 1 from nucleotide 312 to nucleotide 810, or said first 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 SEQ ID No 2 from nucleotide 1 to nucleotide 509.

4. The kit of claim 1, comprising a primer consisting of the sequence of SEQ ID No. 13 and a primer consisting of the sequence of SEQ ID No. 15.

5. A primer pair comprising a first primer having a sequence which, under optimized PCR conditions specifically recognizes a sequence within the 5′

flanking region of A5547-127, and a second primer which, under optimized PCR conditions specifically recognizes a sequence within the foreign DNA contiguous with said 5′

flanking region, or comprising a first primer which, under optimized PCR conditions specifically recognizes a sequence within the 3′

flanking region of A5547-127, and a second primer which, under optimized PCR conditions specifically recognizes a sequence within the foreign DNA contiguous with said 3′

flanking region, wherein said 5′

flanking region comprises 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, said foreign DNA contiguous with said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509.

6. The primer pair of claim 5, wherein one of said primers 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.

7. The primer pair of claim 5, wherein one of said primers 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.

8. A primer pair comprising a first primer comprising at its extreme 3′

end the sequence of SEQ ID No. 13, and a second primer comprising at its extreme 3′

end the sequence of SEQ ID No. 15.

9. A kit comprising a specific probe, capable of hybridizing specifically to a specific region of A5547-127, wherein said specific region is a DNA comprising part of the 5′

flanking sequence of A5547-127 and the sequence of the foreign DNA contiguous therewith, or is a DNA comprising part of the 3′

flanking sequence of A5547-127 and the sequence of the foreign DNA contiguous therewith,

wherein said 5′

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

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, wherein said foreign DNA contiguous with said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509, or the complement of said sequences, and

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.

10. A specific probe comprising a sequence which has at least 80% sequence identity with a sequence comprising part of the 5′

or 3′

flanking sequence of A5547-127 and the sequence of the foreign DNA contiguous therewith, or the complement thereof,

wherein said 5′

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

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 510 to nucleotide 1880, wherein said foreign DNA contiguous with said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509, or the complement of said sequences,

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.

11. A specific probe comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 260 to 360 or of SEQ ID No. 2 from nucleotide 460 to 560, or the complement of said sequences.

12. A DNA comprising the 5′

flanking region of the A5547-127 event and the foreign DNA contiguous therewith, wherein said DNA comprises 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 comprising 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;

wherein said DNA comprises the sequence of SEQ ID No. 1 from nucleotide 260 to nucleotide 360.

13. A DNA comprising the 3′

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

wherein said DNA comprises a sequence 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 foreign DNA contiguous therewith comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 509;

wherein said DNA contains the nucleotide sequence of SEQ ID No. 1 from nucleotide position 460 to nucleotide position 560.

14. An isolated DNA comprising a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence of SEQ ID No 1 from nucleotide 260 to nucleotide 360.

15. An isolated DNA comprising a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence of SEQ ID No 2 from nucleotide 460 to nucleotide 560.

16. An isolated DNA comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 260 to nucleotide 360.

17. An isolated DNA comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 460 to nucleotide 560.