EE-GH3 Cotton Event Identification Using PCR Primers
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Solution Overview
Problem
Current methods for identifying elite transgenic cotton events, such as EE-GH3, are cumbersome and require extensive laboratory setups, making them inefficient for quick and simple identification, especially in distinguishing between transgenic and non-transgenic materials, and ensuring the purity and segregation of GMO and non-GMO products.
Innovation Solution
A method and kit using PCR primers and probes specific to the 5' and 3' flanking regions of the EE-GH3 event, along with a modified epsps gene, to amplify a 334 bp DNA fragment, allowing for the unequivocal identification of the elite event in cotton plants, seeds, and progeny, ensuring genetic stability and agronomic performance equivalent to non-transgenic lines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If current methods for identifying elite transgenic cotton events are used, then identification can be performed, but the process is cumbersome and requires extensive laboratory setups
Solution Approach 1:
The identification system is segmented into specific functional components: event-specific primers (recognizing unique integration sites), confirmation primers (verifying transgene presence), and probe sets that target distinct regions of the EE-GH3 event. This segmentation allows the complex identification process to be broken down into manageable, standardized steps that can be performed with routine laboratory equipment
Solution Approach 2:
The patent establishes preliminary identification protocols and criteria before actual event detection is needed. Pre-designed primer sets and probe sequences are prepared in advance, targeting specific flanking regions and unique sequences of the EE-GH3 event. This preliminary preparation eliminates the need for complex ad-hoc laboratory setups during actual identification, as all necessary reagents and protocols are readied beforehand
2Measurement precision
If current identification methods are used, then transgenic and non-transgenic materials can be distinguished, but the process is not quick and simple
Solution Approach 1:
The patent replaces complex mechanical and manual identification procedures with molecular biological techniques. PCR-based detection methods substitute for time-consuming phenotypic observations and extensive laboratory characterizations. The use of specific primers and probes enables direct molecular detection of the EE-GH3 event, providing quick and simple identification while maintaining high precision in distinguishing transgenic from non-transgenic materials
3Adaptability or versatility
If the transgene is introduced at different locations in the genome, then genetic diversity is achieved, but the phenotype is influenced in different ways
Solution Approach 1:
The patent applies local quality by designing identification primers and probes that target specific local regions of the genome where the EE-GH3 event has integrated. The event-specific primers recognize unique sequences at the integration site, while confirmation primers target the transgene itself. This localized targeting ensures reliable detection regardless of the specific genomic location, maintaining phenotype consistency across different genetic backgrounds
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 method enables rapid, precise identification of EE-GH3 in cotton plants, ensuring genetic stability and agronomic performance comparable to non-transgenic cotton, facilitating the segregation of GMO and non-GMO products and maintaining the desired agronomic characteristics under glyphosate treatment.
Implementation Method 1
The method comprises the steps of: (a) isolating genomic DNA from a cotton plant, seed, cell, tissue or other biological sample; and (b) amplifying a 334 bp DNA fragment from the genomic DNA using the polymerase chain reaction (PCR) and a forward primer having the nucleotide sequence of SEQ ID NO:3 and a reverse primer having the nucleotide sequence of SEQ ID NO:11
Implementation Method 2
a forward primer having the nucleotide sequence of SEQ ID NO:3 and a reverse primer having the nucleotide sequence of SEQ ID NO:11
Data Source
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AI summary
The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.