Elite event EE-GM3 and methods and kits for identifying such event in biological samples

• US 9,631,202 B2
• Filed: 11/23/2010
• Issued: 04/25/2017
• Est. Priority Date: 11/23/2009
• Status: Active Grant

• Current Assignee: BASF Agricultural Solutions Seed US LLC (BASF SE), MS Technologies LLC
• Original Assignee: Bayer Cropscience NV (Bayer AG), MS Technologies LLC
• Inventors: Mason, Justin Thomas, Lettow, Leslie James, Eby, Mark Alan, Eby, William H., Welz, Gnter, Verhaeghe, Steven, De Beuckeleer, Marc, Habex, Veerle, Ferrulo, Jean-Marc
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/511,629
• Publication Number:
  US 20120304330A1
• Time in Patent Office: 2,345 Days
• Field of Search: 435 612, 435 912, 536 2433
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8275 Glyphosate
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides specific transgenic soybean plants, plant material and seeds, characterized in that these products harbor a specific herbicide tolerance transformation event at a specific location in the soybean genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.

Claims

1. A method for identifying elite event EE-GM3 in biological samples, which method comprises detection of an EE-GM3 specific region with a specific primer pair or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the inserted foreign DNA contiguous therewith;

reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

2. The method of claim 1, said method comprising amplifying a DNA fragment of between 50 and 1000 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 foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of SEQ ID No. 1 or its complement, or comprising the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

3. The method of claim 2, wherein said primer recognizing the 5′

flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240, or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

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. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 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. 3 from nucleotide 241 to nucleotide 1408, 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. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

5. The method of claim 4, wherein said primers comprise the sequence of SEQ ID No. 5 and SEQ ID No. 4, respectively, or the sequence of SEQ ID No. 5 and SEQ ID No. 7 respectively.

6. The method of claim 5, which method comprises amplifying a fragment of about 263 or 706 bp using the EE-GM3 PCR identification protocol.

16. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with a specific probe for EE-GM3.

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 EE-GM3 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. 2 from nucleotide 1431 to 1472 or SEQ ID No. 3 from nucleotide 220 to 261, or the complement of said sequences.

7. A kit comprising one primer recognizing the 5′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or one primer recognizing the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and one primer recognizing a sequence within the foreign DNA, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement, wherein

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3;

or

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

8. The kit of claim 7, wherein said primer recognizing the 5′

flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240, or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

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. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 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. 3 from nucleotide 241 to nucleotide 1408, 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. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240, or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

10. The kit of claim 7, comprising a primer comprising the sequence of SEQ ID No. 4 and a primer comprising the sequence of SEQ ID No. 5 or comprising a primer comprising the sequence of SEQ ID No. 5 and a primer comprising the sequence of SEQ ID No. 7.

27. The kit of claim 7, wherein at least one of said primers is labeled.

28. The kit of claim 8, wherein at least one of said primers is labeled.

29. The kit of claim 9, wherein at least one of said primers is labeled.

30. The kit of claim 10, wherein at least one of said primers is labeled.

36. The kit of claim 7, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

37. The kit of claim 8, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

38. The kit of claim 9, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

39. The kit of claim 10, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

45. The kit of claim 7, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

46. The kit of claim 8, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

47. The kit of claim 9, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

48. The kit of claim 10, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

54. The kit of claim 7, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

55. The kit of claim 8, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

56. The kit of claim 9, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

57. The kit of claim 10, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

11. A pair of primers suitable for use in an EE-GM3 specific detection, comprising a first primer which, under optimized detection conditions specifically recognizes a sequence within the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 and said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and a second primer recognizing a sequence within the foreign DNA contiguous with said 5′

or 3′

flanking region in EE-GM3, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement, wherein

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3;

or

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

12. The primer pair of claim 11, wherein said first primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said second primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240.

13. The primer pair of claim 11, wherein said first 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. 2 from nucleotide 1 to nucleotide 1451 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408.

14. The primer pair of claim 11, wherein said first primer comprises at its extreme 3′

end the sequence of SEQ ID No. 5.

31. The primer pair of claim 11, wherein at least one of said primers is labeled.

32. The primer pair of claim 12, wherein at least one of said primers is labeled.

33. The primer pair of claim 13, wherein at least one of said primers is labeled.

34. The primer pair of claim 14, wherein at least one of said primers is labeled.

40. The primer pair of claim 11, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

41. The primer pair of claim 12, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

42. The primer pair of claim 13, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

43. The primer pair of claim 14, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

49. The primer pair of claim 11, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

50. The primer pair of claim 12, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

51. The primer pair of claim 13, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

52. The primer pair of claim 14, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

58. The primer pair of claim 11, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

59. The primer pair of claim 12, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

60. The primer pair of claim 13, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

61. The primer pair of claim 14, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

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

end the sequence of SEQ ID No. 5 and a second primer comprising at its extreme 3′

end the sequence of SEQ ID No. 4, wherein

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3;

or

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

35. The primer pair of claim 15, wherein at least one of said primers is labeled.

44. The primer pair of claim 15, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

53. The primer pair of claim 15, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

62. The primer pair of claim 15, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

19. A kit comprising a specific probe, capable of hybridizing specifically to a specific region of EE-GM3, 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 foreign DNA comprising herbicide tolerance genes in EE-GM3 and the sequence of the foreign DNA contiguous therewith;

reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

20. The kit of claim 19, wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences.

21. A specific probe for the identification of elite event EE-GM3 in biological samples, which comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the sequence of the foreign DNA contiguous therewith, or the complement thereof;

reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

22. The probe of claim 21 which has at least 80% sequence identity with SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences.

23. A specific probe comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences.

24. A method for confirming seed purity, which method comprises detection of an EE-GM3 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the inserted, foreign DNA contiguous therewith, in seed samples;

reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

25. A method for screening seeds for the presence of EE-GM3, which method comprises detection of an EE-GM3 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the inserted, foreign DNA contiguous therewith, in samples of seed lots;

reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

26. A method of detecting the presence of elite event EE-GM3 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:

target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising;

a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1469 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide 223 to nucleotide 240 or its complement;

b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1434 to nucleotide 1451 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide 241 to nucleotide 258 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;

c) cleaving only the labeled probe within the probe;

target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;

d) recycling of the target nucleic acid sequence by repeating steps (a) to (c); and

e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.