DNA DETECTION METHODS FOR SITE SPECIFIC NUCLEASE ACTIVITY

• US 20140298547A1
• Filed: 12/13/2013
• Published: 10/02/2014
• Est. Priority Date: 12/13/2012
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Inventors: Sastry-Dent, Lakshmi, Cao, Zehui, Chen, Wei, Zhou, Ning, Webb, Steven R., Simpson, Matthew
• Granted Patent: US 9,493,844 B2
• Time in Patent Office: Days
• US Class Current: 800/306
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

The present disclosure provides methods for detecting and identifying plant events that contain precision targeted genomic loci, and plants and plant cells comprising such targeted genomic loci. The method can be deployed as a high throughput process utilized for screening the intactness or disruption of a targeted genomic loci and optionally for detecting a donor DNA polynucleotide insertion at the targeted genomic loci. The methods are readily applicable for the identification of plant events produced via a targeting method which results from the use of a site specific nuclease.

Claims

1. A method for identifying the presence of an exogenous donor DNA polynucleotide inserted within a targeted genomic locus comprising:

a. amplifying in a first amplification reaction a genomic DNA sample comprising the targeted genomic locus using a first plurality of oligonucleotides that bind under hybridization conditions proximal to the targeted genomic locus, to thereby generate a first amplicon comprising the targeted genomic locus; and

,

b. detecting the presence or absence of the first amplicon, wherein the absence of the first amplicon indicates the presence of the donor DNA polynucleotide within the targeted genomic locus.

2. The method of claim 1, the method further comprising:

a. amplifying in a second amplification reaction the genomic DNA sample using a second plurality of oligonucleotides that bind under hybridization conditions proximal to the targeted genomic locus and within the donor DNA polynucleotide, to thereby generate a second amplicon comprising at least a portion of the targeted genomic locus and at least a portion of the donor DNA polynucleotide; and

,

b. detecting the presence or absence of the second amplicon, wherein the presence of the second amplicon indicates the presence of the donor DNA polynucleotide within the targeted genomic locus.

3. The method of claim 2, the method further comprising:

a. quantitating the results of the first amplification reaction;

b. quantitating the results of the second amplification reaction;

c. comparing the results of the first and second amplification reactions; and

,

d. determining the presence or absence of the donor DNA polynucleotide within the targeted genomic locus, wherein the donor DNA polynucleotide is confirmed as inserted within the targeted genomic locus if the first amplicon is absent and the second amplicon is present.

4. The method of claim 3, the method further comprising a multiplex reaction, wherein the first and second amplification reactions are run in a single tube or well.

5. The method of claim 3, wherein quantitating the results of the of first and second amplification reactions comprises producing a signature profile for one or both of the first and second amplification reactions.

6. The signature profile of claim 5, wherein the signature profile is selected from the group consisting of a melting temperature curve signature profile and a fluorescence signature profile.

7. The method of claim 5, wherein the signature profile is produced from an intercalating DNA dye.

8. The method of claim 7, wherein the intercalating DNA dye comprises a cyanine dye.

9. The method of claim 8, wherein the cyanine dye is used in an amplification reaction at a concentration of less than 4 μ

M.

10. The method of claim 8, wherein the cyanine dye is used in an amplification reaction at a concentration of less than 2.7 μ

M.

11. The method of claim 5, wherein the signature profile is produced from a fluorescent dye.

12. The method of claim 1, the method further comprising:

a. selecting a transgenic event comprising the donor DNA polynucleotide within the targeted genomic locus.

13. A plant, comprising the transgenic event of claim 12.

14. The plant of claim 13, wherein the plant is a dicot plant.

15. The dicot plant of claim 14, wherein the dicot plant is selected from the group consisting of a soybean plant, a canola plant and a cotton plant.

16. The plant of claim 13, wherein the plant is a monocot plant.

18. The method of claim 1, wherein the genomic locus is cleaved by a site specific nuclease.

19. The method of claim 18, wherein the nuclease comprises a zinc finger nuclease.

20. The method of claim 18, wherein the nuclease comprises a TALEN or CRISPR nuclease.

21. The method of claim 1, wherein the amplifying comprises amplifying in a polymerase chain reaction.

22. The method of claim 2, wherein the second amplicon comprises a 5′

junction of the donor DNA polynucleotide and the targeted genomic locus.

23. The method of claim 2, wherein the second amplicon comprises a 3′

junction of a donor DNA polynucleotide and the targeted genomic locus.

24. The method of claim 1, wherein the donor DNA polynucleotide comprises at least one gene expression cassette.

25. The method of claim 1 or claim 2, wherein the first or second plurality of oligonucleotides, or both, comprise a fluorescent dye.

26. The method of claim 25, wherein the fluorescent dye is selected from the group consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

17. The monocot plant of claim 17, wherein the monocot plant is selected from the group consisting of a corn plant, a rice plant, and a wheat plant.

27. A method for identifying a disruption of a genomic locus comprising:

a. amplifying in a first amplification reaction a genomic DNA sample comprising the disrupted genomic locus using a plurality of oligonucleotides that bind under hybridization conditions proximal to the disrupted genomic locus, to thereby generate a first amplicon comprising the disrupted genomic locus; and

,

b. detecting the presence or absence of the first amplicon, wherein the absence of the amplicon indicates the disruption of the genomic locus.

28. The method of claim 27, the method further comprising:

a. identifying the presence of a donor insertion within a disrupted genomic locus; and

,

b. selecting a transgenic event comprising a donor insertion within a disrupted genomic locus.

29. A plant, comprising the transgenic event of claim 28.

30. The plant of claim 29, wherein the plant is a dicot plant.

31. The dicot plant of claim 30, wherein the dicot plant is selected from the group consisting of a soybean plant, a canola plant and a cotton plant.

32. The plant of claim 29, wherein the plant is a monocot plant.

33. The monocot plant of claim 32, wherein the monocot plant is selected from the group consisting of a corn plant, a rice plant, and a wheat plant.

34. The method of claim 27, wherein the genomic locus is cleaved by a site specific nuclease.

35. The method of claim 34, wherein the nuclease comprises a zinc finger nuclease.

36. The method of claim 34, wherein the nuclease comprises a TALEN or CRISPR nuclease.

37. The method of claim 27, wherein the amplifying comprises amplifying in a polymerase chain reaction.

38. The method of claim 27, wherein the donor DNA polynucleotide comprises at least one gene expression cassette.

39. The method of claim 27, wherein the plurality of oligonucleotides, or both, comprise a fluorescent dye.

40. The method of claim 39, wherein the fluorescent dye is selected from the group consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

41. A method for identifying a disruption of a genomic locus from a plurality of plant cells comprising:

a. amplifying in a first amplification reaction a genomic DNA sample comprising the disrupted genomic locus using a plurality of oligonucleotides that bind under hybridization conditions proximal to the disrupted genomic locus, to thereby generate a first amplicon comprising the disrupted genomic locus;

b. quantitating the results of the first amplification reaction;

c. amplifying in a second amplification reaction a genomic DNA sample comprising the disrupted genomic locus using the plurality of oligonucleotides that bind under hybridization conditions proximal to the disrupted genomic locus, to thereby generate a second amplicon comprising the disrupted genomic locus;

d. quantitating the results of the second amplification reaction; and

,

e. comparing the quantity of the first and second amplification reactions, wherein the quantity of the first amplification reaction comprises a lower quantity of amplified product as compared to the second amplification reaction thereby indicating the disruption of a genomic locus in the first amplicon samples.

42. The method of claim 41, wherein quantitating the results of the of first and second amplification reactions comprises producing a signature profile for one or both of the first and second amplification reactions.

43. The signature profile of claim 42, wherein the signature profile is selected from the group consisting of a melting temperature curve signature profile and a fluorescence signature profile.

44. The method of claim 43, wherein the signature profile is produced from an intercalating DNA dye.

45. The method of claim 44, wherein the intercalating DNA dye comprises a cyanine dye.

46. The method of claim 45, wherein the cyanine dye is used in an amplification reaction at a concentration of less than 4 μ

M.

47. The method of claim 45, wherein the cyanine dye is used in an amplification reaction at a concentration of less than 2.7 μ

M.

48. The method of claim 42, wherein the signature profile is produced from a fluorescent dye.

49. The method of claim 41, the method further comprising:

a. identifying the presence of a donor insertion within a targeted genomic locus; and

,

b. selecting a transgenic event comprising a donor insertion within a targeted genomic locus.

50. A plant, comprising the transgenic event of claim 49.

51. The plant of claim 50, wherein the plant is a dicot plant.

52. The dicot plant of claim 51, wherein the dicot plant is selected from the group consisting of a soybean plant, a canola plant and a cotton plant.

53. The plant of claim 50, wherein the plant is a monocot plant.

54. The monocot plant of claim 53, wherein the monocot plant is selected from the group consisting of a corn plant, a rice plant, and wheat plant.

55. The method of claim 41, wherein the genomic locus is cleaved by a site specific nuclease.

56. The method of claim 55, wherein the nuclease comprises a zinc finger nuclease.

57. The method of claim 55, wherein the nuclease comprises a TALEN or CRISPR nuclease.

58. The method of claim 41, wherein the amplifying comprises amplifying in a polymerase chain reaction.

59. The method of claim 41, wherein the donor DNA polynucleotide comprises at least one gene expression cassette.

60. The method of claim 41, wherein the plurality of oligonucleotides comprise a fluorescent dye.

61. The method of claim 60, wherein the fluorescent dye is selected from the group consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.