Wheat Endogenous DNA Detection via Proline-Rich Protein Gene Primers
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Solution Overview
Problem
Current methods for detecting and quantitating endogenous wheat DNA are hindered by the high homology of wheat with other cereal grains, leading to false detection and variability across wheat varieties, making it difficult to accurately identify and measure wheat DNA in food samples without cross-reactivity.
Innovation Solution
A method utilizing a partial region of the proline-rich protein (PRP) gene, specifically amplifying a nucleotide sequence of at least 80 bases, using primer pairs that do not cross-react with other crops, allowing for specific detection and quantitation of endogenous wheat DNA through PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR methods using general cereal grain primers are used, then detection of wheat DNA can be performed, but false detection occurs due to high homology with other cereal grains
Solution Approach 1:
The patent applies local quality by selecting a specific gene region (proline-rich protein gene) that has unique sequence characteristics in wheat compared to other cereal grains. Instead of using general cereal grain primers that cross-react, the invention targets a localized specific region with high sequence divergence, allowing specific amplification of wheat DNA while avoiding false detection from homologous sequences in other grains.
Solution Approach 2:
The patent segments the wheat genome by focusing on a specific gene family (proline-rich protein genes) rather than attempting to detect all wheat DNA sequences. By dividing the complex genome into specific detectable regions with unique characteristics, the method achieves specific identification of wheat among cereal grains through targeted amplification of these segmented regions.
2Measurement precision
If PCR amplification is used to detect endogenous wheat DNA, then quantitation can be performed, but variability across wheat varieties affects measurement precision
Solution Approach 1:
The patent achieves universality by designing primers that target conserved regions within the proline-rich protein gene family, which are present across multiple wheat varieties. The method can detect and quantitate wheat DNA from different varieties using the same primer pair, making the assay universally applicable while maintaining measurement precision through targeted amplification of conserved sequences.
Solution Approach 2:
The patent applies parameter changes by optimizing the primer binding region to target sequences with appropriate levels of conservation. By selecting a specific region within the proline-rich protein gene that balances conservation (for variety compatibility) and specificity (for accurate quantitation), the method achieves both measurement precision and adaptability across wheat varieties.
3Measurement precision
If standard reference materials are used for quantitative PCR, then contamination rate can be determined, but the process becomes complex when multiple GMO strains are present
Solution Approach 1:
The patent extracts the endogenous wheat DNA signal as a separate reference component, distinct from the GMO-specific transgenic sequences. By using the proline-rich protein gene as an internal reference that is always present in wheat but absent from transgenic insertions, the method simplifies multi-strain analysis by providing a universal baseline for quantitation that works across different GMO strains without requiring separate standard reference materials for each strain.
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 precise detection and quantitation of endogenous wheat DNA in food samples, reducing false positives and variability, and accurately determining the contamination rate of genetically modified wheat strains.
Implementation Method 1
specifically amplifying a nucleotide sequence of at least 80 bases, using primer pairs that do not cross-react with other crops, allowing for specific detection and quantitation of endogenous wheat DNA through PCR
Data Source
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AI summary
An object of the present invention is to discover an endogenous wheat sequence satisfying the conditions of: a) it is universally present in varieties of wheat, b) the amount present (detected amount) is not affected depending on the wheat variety, c) even if other grains are present, only wheat can be detected without cross-reactivity, and d) it is amplified quantitatively by the PCR reaction. A further object of the present invention is to provide a method of accurately detecting and quantitating endogenous wheat DNA in a test sample by the polymerase chain reaction. The present invention provides a method of detecting or quantitating endogenous wheat DNA in a test sample by the polymerase chain reaction, the method comprising: a step of using a nucleic acid in the test sample or a nucleic acid extracted from the test sample as a template to amplify the nucleic acid of a region consisting of the base sequence identified as SEQ ID NO: 2 or a partial sequence thereof with a primer pair capable of amplifying that region; and a step of detecting or quantitating the amplified nucleic acid.