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

• US 8,017,756 B2
• Filed: 04/04/2006
• Issued: 09/13/2011
• 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
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US11/911,167
• Publication Number:
  US 20080196127A1
• Time in Patent Office: 1,988 Days
• Field of Search: None
• US Class Current: 536/24.3
• 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, comprising detecting a A5547-127 specific region with a specific primer combination or probe which specifically recognizes part of the 5′

or 3′

flanking region of A5547-127 and part of the foreign DNA contiguous therewith, wherein said specific region comprises part of the 5′

or 3′

flanking region of said elite event and part of the foreign DNA contiguous therewith.

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 1 to nucleotide 509.

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 1 to nucleotide 509.

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 1 to nucleotide 509.

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.

7. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with said specific probe.

8. The method of claim 7, 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.

9. The method of claim 8, 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.

12. The method of claim 1, wherein said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 510 to nucleotide 1880 or the complement thereof, and wherein said foreign DNA comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 312 to nucleotide 810 or the complement thereof, or comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 509 or the complement thereof.

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

or 3′

flanking region of A5547-127 and part of the foreign DNA contiguous therewith, in seed samples, wherein said specific region comprises part of the 5′

or 3′

flanking region of A5547-127 and part of the foreign DNA contiguous therewith.

13. The method of claim 10, wherein said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 510 to nucleotide 1880 or the complement thereof, and wherein said foreign DNA comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 312 to nucleotide 810 or the complement thereof, or comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 509 or the complement thereof.

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

or 3′

flanking region of A5547-127 and part of the foreign DNA contiguous therewith, in samples of seed lots, wherein said specific region comprises part of the 5′

or 3′

flanking region of A5547-127 and part of the foreign DNA contiguous therewith.

14. The method of claim 11, wherein said 5′

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

flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 510 to nucleotide 1880 or the complement thereof, and wherein said foreign DNA comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 312 to nucleotide 810 or the complement thereof, or comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 509 or the complement thereof.