Maize DP-004114-3 Detection via Flanking DNA Sequences
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Solution Overview
Problem
Current methods for detecting specific transgenic events in crops, such as corn event DP-004114-3, are not discriminative enough, particularly when using DNA constructs like those from Bacillus thuringiensis, as they often rely on interchangeable coding regions and may not differentiate between similar constructs without knowing the flanking DNA sequence, making it difficult to identify optimal expression levels and patterns for insect resistance.
Innovation Solution
A DNA construct comprising the cry1F, cry34Ab1, cry35Ab1, and pat gene cassettes, integrated into maize using Agrobacterium-mediated transformation, which confers resistance to lepidopteran and coleopteran pests, along with specific primers and probes that recognize unique flanking sequences of DP-004114-3 for precise identification in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA constructs from Bacillus thuringiensis are used for insect resistance, then insect resistance is achieved, but detection precision is insufficient due to interchangeable coding regions
Solution Approach 1:
The patent uses flanking DNA sequences as intermediary markers to indirectly detect the presence of Bt transgenes. Instead of attempting to detect the interchangeable coding regions directly, the method detects the unique flanking sequences that border the transgene insertion site, which serve as reliable proxies for identifying the specific transgenic event.
Solution Approach 2:
The patent creates specific PCR primers and probes that copy or replicate the unique flanking sequence patterns associated with each transgenic event. These copied sequences enable discrimination between different transgenic events by matching against known flanking sequence signatures from the transformation process.
2Reliability
If multiple transgenic events are screened to identify optimal expression, then expression optimization is achieved, but detection and identification become more difficult
Solution Approach 1:
The patent segments the detection approach into two distinct parts: detecting the flanking DNA sequences that are unique to each transformation event, and separately analyzing the transgene expression levels. This segmentation allows each aspect to be optimized independently - the flanking sequences provide event-specific identification while expression analysis determines functional performance.
Solution Approach 2:
The patent applies local quality by focusing detection efforts on the specific flanking regions surrounding each transgene insertion site rather than attempting to detect the entire transgene construct. These local flanking sequences have unique characteristics that serve as fingerprints for each transformation event, enabling precise identification without needing to analyze the entire interchangeable coding region.
3Measurement precision
If flanking DNA sequence information is required for detection, then detection precision improves, but ease of operation decreases due to additional sequencing requirements
Solution Approach 1:
The patent performs preliminary action by characterizing and storing the flanking DNA sequences during the initial transformation and event selection process. Once these flanking sequences are identified and recorded, they can be used to design specific PCR primers and probes in advance, eliminating the need for repeated sequencing operations during routine detection and allowing direct amplification-based assays.
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
The solution provides effective insect resistance to targeted pests and enables reliable detection of the DP-004114-3 event through specific PCR assays and hybridization methods, ensuring compliance with regulations and quality control in crop products.
Implementation Method 1
integrated into maize using Agrobacterium-mediated transformation
Implementation Method 2
specific PCR assays
Implementation Method 3
hybridization methods
Data Source
AI summary
The invention provides DNA compositions that relate to transgenic insect resistant maize plants. Also provided are assays for detecting the presence of the maize DP-004114-3 event based on the DNA sequence of the recombinant construct inserted into the maize genome and the DNA sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.