DNA detection methods for site specific nuclease activity

Patent metadata

Field	Value
Publication number	US-10344322-B2
Application number	US-201615249684-A
Country	US
Kind code	B2
Filing date	Aug 29, 2016
Priority date	Dec 13, 2012
Publication date	Jul 9, 2019
Grant date	Jul 9, 2019

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.

First claim

What is claimed is:

1. 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 to the disrupted genomic locus, wherein at least one oligonucleotide is a primer designed to bind the disrupted genomic locus, to thereby generate a first amplicon comprising the disrupted genomic locus; and,
b. detecting the absence of the first amplicon, wherein the absence of the amplicon indicates the disruption of the genomic locus.

2. The method of claim 1 , 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.

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

4. The method of claim 3 , wherein the nuclease comprises a zinc finger nuclease.

5. The method of claim 3 , wherein the nuclease comprises a TALEN or CRISPR nuclease.

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

7. The method of claim 2 , wherein the donor DNA polynucleotide comprises at least one gene expression cassette.

8. The method of claim 1 , wherein the plurality of oligonucleotides comprise a fluorescent dye.

9. The method of claim 8 , 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.

10. 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 to the disrupted genomic locus, wherein at least one oligonucleotide is a primer designed to bind 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.

11. The method of claim 10 , 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.

12. The method of claim 11 , wherein the signature profile is produced from an intercalating DNA dye.

13. The method of claim 12 , wherein the intercalating DNA dye comprises a cyanine dye.

14. The method of claim 13 , wherein the cyanine dye is used in an amplification reaction at a concentration of less than 4 μM.

15. The method of claim 13 , wherein the cyanine dye is used in an amplification reaction at a concentration of less than 2.7 μM.

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

17. The method of claim 10 , 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.

18. The method of claim 10 , 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 10 , wherein the amplifying comprises amplifying in a polymerase chain reaction.

22. The method of claim 10 , wherein the donor DNA polynucleotide comprises at least one gene expression cassette.

23. The method of claim 10 , wherein the plurality of oligonucleotides comprise a fluorescent dye.

24. The method of claim 23 , 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.

Assignees

• Dow Agrosciences Llc

Inventors

• Sastry-Dent Lakshmi
• Simpson Matthew A
• Cao Zehui
• Chen Wei
• Zhou Ning
• Webb Steven R

Classifications

• C12N5/00

  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 title

• A01G24/00

  Growth substrates; Culture media; Apparatus or methods therefor (for mushrooms A01G18/20) · CPC title

• C12Q1/6895 Primary

  for plants, fungi or algae · CPC title

• C12Q2600/13

  Plant traits · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title