Rapid Targeting Assay in Crops for Determining Donor Insertion

摘要

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 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.

主权项

1. A method for detecting site specific integration of a polynucleotide donor sequence within a genomic target site, the method comprising:
amplifying a genomic DNA with a first round of PCR to produce a first amplicon using a first Out-PCR primer designed to bind to the genomic DNA target site and a first In-PCR primer designed to bind the integrated polynucleotide donor sequence; amplifying the first amplicon with a second round of PCR using primers specific to sequences located within the first amplicon to produce a second amplicon; and, detecting the presence of the second amplicon, wherein the production of the second amplicon indicates the presence of the site specific integration event.