DAS-59132 Detection via Flanking Sequence PCR
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Solution Overview
Problem
Current methods for detecting specific maize events, such as DAS-59132, are not discriminative enough, particularly when using DNA constructs that are similar or identical, making it difficult to distinguish between different events and ensuring compliance with regulations and quality control in food and environmental monitoring.
Innovation Solution
Development of event-specific PCR assays and DNA probes that target the unique 3' flanking sequence of maize event DAS-59132, allowing for the identification of the transgene and flanking insertion region, using primer sequences and probes that produce unique amplicons diagnostic for the event.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If general transgene detection methods are used, then detection capability is achieved, but discrimination between different maize events is insufficient
Solution Approach 1:
The detection method is segmented into two distinct parts: a common primer that binds to conserved transgene sequences and an event-specific primer that binds to unique flanking sequences. This segmentation allows the assay to first identify the presence of any transgene and then specifically distinguish the DAS-59132 event, resolving the contradiction between general detection capability and event-specific discrimination.
Solution Approach 2:
The invention applies local quality by designing the event-specific primer to target only the unique 3' flanking region of DAS-59132, while the common primer targets conserved regions. This localized specificity at the molecular level enables high discrimination precision without requiring complete redesign of the entire detection system, thus managing complexity while improving measurement precision.
2Measurement precision
If event-specific detection is implemented, then discrimination accuracy is improved, but detection method complexity increases
Solution Approach 1:
The common primer serves multiple functions: it detects the presence of transgenes in general, provides a binding site for PCR amplification, and works across different maize events that share the same transgene construct. This multi-functionality reduces the need for completely separate assays for each event, thereby limiting the increase in overall system complexity while maintaining high event identification accuracy through the complementary event-specific primer.
3Measurement precision
If flanking sequence targeting is used, then event specificity is enhanced, but detection reliability may be affected by genomic variation
Solution Approach 1:
The invention changes the detection parameter from targeting highly variable regions to targeting the 3' flanking sequence, which exhibits sufficient conservation within the DAS-59132 event population. This parameter change maintains event specificity while improving detection consistency, as the flanking sequence shows less variation compared to other potential target regions, thereby resolving the contradiction between specificity and reliability.
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
Enables accurate and discriminative detection of maize event DAS-59132 in plant materials and products, ensuring compliance with regulations and quality control by producing event-specific amplicons that are unique to the target sequence.
Implementation Method 1
contacting the sample with a DNA primer set, that when used in a nucleic acid amplification reaction produces an amplicon that is diagnostic for the 3' junction of corn event DAS-59132; performing a nucleic acid amplification reaction, thereby producing the amplicon
Implementation Method 2
contacting the sample comprising DNA with a DNA probe, molecule that hybridizes under stringent hybridization conditions with DNA extracted from corn event DAS-59132
Data Source
AI summary
The invention provides assays for detecting the presence of the maize DAS-59132 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.