DNA detection methods for site specific nuclease activity

Patent metadata

Field	Value
Publication number	US-9493844-B2
Application number	US-201314105223-A
Country	US
Kind code	B2
Filing date	Dec 13, 2013
Priority date	Dec 13, 2012
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

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 the presence of an exogenous donor DNA polynucleotide inserted within a single targeted eukaryotic genomic locus, the method comprising the following steps:
a. cleaving the single eukaryotic genomic locus with a site specific nuclease to yield a cleaved eukaryotic genomic locus;
b. inserting the exogenous donor DNA within the cleaved eukaryotic genomic locus;
c. amplifying in a first amplification reaction a genomic DNA sample comprising the targeted eukaryotic genomic locus using a first plurality of primers that bind under hybridization conditions proximal to the targeted eukaryotic genomic locus, wherein at least one of the primers binds to the site specific nuclease cleavage site, to thereby generate a first amplicon comprising the targeted eukaryotic genomic locus;
d. detecting the presence or absence of the first amplicon, wherein the absence of the first amplicon indicates the presence of the exogenous donor DNA polynucleotide within the targeted eukaryotic genomic locus;
e. amplifying in a second amplification reaction the genomic DNA sample using a second plurality of primers that bind under hybridization conditions proximal to the targeted eukaryotic genomic locus and within the exogenous donor DNA polynucleotide, to thereby generate a second amplicon comprising at least a portion of the targeted eukaryotic genomic locus and at least a portion of the exogenous donor DNA polynucleotide; and,
f. detecting the presence or absence of the second amplicon, wherein the presence of the second amplicon indicates the presence of the exogenous donor DNA polynucleotide within the targeted eukaryotic genomic locus.

2. The method of claim 1 , 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 exogenous donor DNA polynucleotide within the targeted eukaryotic genomic locus, wherein the exogenous donor DNA polynucleotide is confirmed as inserted within the targeted eukaryotic genomic locus if the first amplicon is absent and the second amplicon is present.

3. The method of claim 2 , the method further comprising a multiplex reaction, wherein the first and second amplification reactions are run in a single tube or well.

4. The method of claim 2 , 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.

5. The signature profile of claim 4 , wherein the signature profile is selected from the group consisting of a melting temperature curve signature profile and a fluorescence signature profile.

6. The method of claim 4 , wherein the signature profile is produced from an intercalating DNA dye.

7. The method of claim 6 , wherein the intercalating DNA dye comprises a cyanine dye.

8. The method of claim 7 , wherein the cyanine dye is used in an amplification reaction at a concentration of less than 4 μM.

9. The method of claim 7 , wherein the cyanine dye is used in an amplification reaction at a concentration of less than 2.7 μM.

10. The method of claim 4 , wherein the signature profile is produced from a fluorescent dye.

11. The method of claim 1 , the method further comprising:
a. selecting a transgenic event comprising the exogenous donor DNA polynucleotide within the targeted eukaryotic genomic locus.

12. The method of claim 1 , wherein the nuclease comprises a zinc finger nuclease.

13. The method of claim 1 , wherein the nuclease comprises a TALEN or CRISPR nuclease.

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

15. The method of claim 1 , wherein the second amplicon comprises a 5′ junction of the exogenous donor DNA polynucleotide and the targeted eukaryotic genomic locus.

16. The method of claim 1 , wherein the second amplicon comprises a 3′ junction of the exogenous donor DNA polynucleotide and the targeted eukaryotic genomic locus.

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

18. The method of claim 1 , wherein the first or second plurality of primers, or both, comprise a fluorescent dye.

19. The method of claim 18 , 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.

20. The method of claim 1 , wherein the primers further comprise a probe.

Assignees

• Dow Agrosciences Llc

Inventors

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

Classifications

• C12Q1/6895 Primary

  for plants, fungi or algae · CPC title

• C12Q2600/13

  Plant traits · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title