DNA Detection for Site-Specific Nuclease Activity
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Solution Overview
Problem
Current methods for targeted genome modification in plants are inefficient, requiring the screening of large numbers of plant events to identify specific genomic loci with targeted gene insertion, and often lead to inaccurate estimation due to reliance on single analytical methods.
Innovation Solution
A high-throughput method involving two amplification reactions, a disruption assay and an In-Out PCR amplification reaction, to rapidly identify and confirm the presence of a donor DNA polynucleotide inserted within a targeted genomic locus, utilizing specific oligonucleotides and fluorescent dyes for accurate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current single analytical methods are used for confirming targeting, then the screening process is simple, but the accuracy of targeting frequency estimation is low and confidence outcomes are reduced
Solution Approach 1:
The screening process is divided into two distinct analytical methods: a first amplification reaction to detect disruption of the genomic locus, and a second amplification reaction to detect presence of the donor DNA polynucleotide. This segmentation allows each method to focus on a specific aspect of targeting confirmation, thereby improving overall measurement precision while maintaining manageable complexity through modular design
Solution Approach 2:
The patent combines two complementary analytical methods into a unified screening approach. The first method detects the absence of the original genomic sequence, while the second detects the presence of the inserted donor DNA. By merging these two detection strategies, the system achieves higher confidence outcomes and more accurate targeting frequency estimation than either method could provide alone
2Reliability
If large numbers of plant events are screened to identify targeted genomic loci, then the probability of finding targeted events increases, but the time and resources required for screening increase
Solution Approach 1:
The first amplification reaction serves as a preliminary screening step to quickly identify plant events with disruption of the targeted genomic locus. By performing this initial detection before the second amplification reaction, the system efficiently filters samples and reduces the overall screening time while maintaining high reliability in identifying true targeted events
Solution Approach 2:
The two-step amplification process provides feedback validation: the first reaction identifies potential targeted events, and the second reaction confirms the presence of donor DNA. This feedback mechanism increases the probability of correctly identifying targeted events by eliminating false positives, thereby improving reliability without requiring excessive screening of additional samples
3Manufacturing precision
If site specific nuclease methods are used for targeted genome modification, then precise genomic loci modification is achieved, but the efficiency of targeted gene insertion remains low
Solution Approach 1:
The patent replaces traditional, less efficient detection methods with a highly sensitive and specific dual amplification system. By using PCR-based detection with specifically designed oligonucleotides and fluorescent dyes, the system achieves rapid and accurate identification of targeted events, thereby improving the overall efficiency of the targeted gene insertion process while maintaining the precision of site specific nuclease cutting
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method significantly increases the probability of identifying targeted events by providing a high-quality, high-throughput process for selecting transgenic events with precise donor insertion, allowing for the analysis of large numbers of plant samples and confirming the integrity of genomic loci.
Implementation Method 1
a first plurality of oligonucleotides that bind under hybridization conditions proximal to the targeted genomic locus
Implementation Method 2
utilizing specific oligonucleotides and fluorescent dyes for accurate detection
Data Source
AI summary
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.