TaqMan PCR Assay for AAD-1 Corn Event Detection
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Solution Overview
Problem
Current methods for detecting and analyzing the AAD-1 corn event, such as the Invader assay, are sensitive to DNA quality, require high DNA quantities, and are inefficient for large-scale sample analysis, limiting their flexibility in commercial settings.
Innovation Solution
Development of fluorescence-based endpoint TaqMan PCR assays utilizing the endogenous invertase gene as a reference for high-throughput zygosity analysis, which eliminates the need for a denaturing step and allows for faster processing of large numbers of samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current detection methods (Invader assay) are used, then detection sensitivity is maintained, but DNA quality sensitivity increases and processing efficiency decreases
Solution Approach 1:
The patent changes the detection parameters by using fluorescence-based endpoint TaqMan PCR assays instead of the Invader assay. This involves modifying the chemical and physical parameters of the detection system - using fluorescent probes and PCR amplification conditions that are less sensitive to DNA quality variations, thereby improving reliability while maintaining productivity
Solution Approach 2:
The patent replaces the mechanical/enzymatic system of the Invader assay with a fluorescence-based PCR system. This substitution eliminates the need for certain enzymatic steps and replaces them with a more robust thermal cycling and fluorescence detection system that is less affected by DNA quality issues
2Loss of time
If current detection methods are used, then detection accuracy is maintained, but time consumption increases for large-scale analysis
Solution Approach 1:
The patent performs preliminary PCR amplification of the target DNA sequences before detection. By pre-amplifying the DNA using specific primers and fluorescent probes, the method reduces the time needed for subsequent detection steps while ensuring that sufficient target material is available for accurate measurement, thus reducing time loss without compromising precision
3Ease of operation
If current detection methods are used, then comprehensive analysis is achieved, but operational flexibility decreases in commercial settings
Solution Approach 1:
The patent combines multiple detection functions into a single TaqMan PCR assay system. By integrating amplification, detection, and quantification into one unified fluorescence-based platform, the method improves ease of operation while maintaining the ability to analyze large numbers of samples efficiently, thus enhancing both operational flexibility and productivity
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 TaqMan PCR assays provide efficient and accurate detection of the AAD-1 corn event, offering time savings and improved flexibility for large-scale analysis, while maintaining high specificity and reducing the risk of assay failure due to DNA quality issues.
Implementation Method 1
fluorescence-based endpoint TaqMan PCR assays
Data Source
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AI summary
This invention relates in part to detecting herbicide tolerant plants - more specifically, an aad-1 transformation event in corn plants. The subject invention also provides assays for detecting the presence of the subject event in a sample (of corn grain, for example). Kits and conditions useful in conducting the assays are also provided. The subject invention also relates in part to plant breeding using the subject methods. In some embodiments, said event / polynucleotide sequence can be "stacked" with other traits. More specifically, the invention relates in part to an endpoint TaqMan PCR assay for AAD-1 corn event 40278-9. Some embodiments are directed to assays that are capable of high throughput zygosity analysis. The subject invention further relates, in part, to the use of a preferred reference gene for use in determining zygosity.