Universal Terminator Primer Set for Transgene Quantification
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Solution Overview
Problem
Current methods for quantifying transgene expression and copy number in transgenic plants are cumbersome, prone to errors, and costly, especially when dealing with multiple transgenes, as they require gene-specific primers and probes, and struggle with differentiating between endogenous and transgenic gene expression.
Innovation Solution
The use of real-time PCR with primers and probes specific to the heterologous terminator sequence linked to the transgene allows for high-throughput quantification of transgene expression and copy number, utilizing a universal primer set that hybridizes to the terminator sequence, thereby overcoming the limitations of gene-specific primer sets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If gene-specific primers and probes are used for quantifying transgene expression, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent employs a universal primer set that targets the heterologous terminator sequence present in all transgenic plants, allowing a single primer set to quantify multiple different transgenes. This eliminates the need for developing and maintaining multiple gene-specific primer sets, thereby reducing device complexity while maintaining measurement precision through the conserved terminator sequence target.
2Measurement precision
If multiple gene-specific primer sets are developed for different transgenes, then measurement precision is improved, but loss of time increases
Solution Approach 1:
By designing primers that bind to the heterologous terminator sequence common to all transgenes, the method enables simultaneous detection and quantification of multiple transgenes in a single PCR reaction. This universal approach dramatically reduces the time required for high-throughput screening compared to sequential analysis using multiple gene-specific primer sets.
3Measurement precision
If quantitative PCR with gene-specific primers is used, then measurement precision is improved, but cost increases
Solution Approach 1:
The universal primer set targeting the heterologous terminator sequence allows a single reagent combination to quantify multiple different transgenes, eliminating the need to purchase and use multiple sets of expensive gene-specific primers and probes. This significantly reduces reagent costs while maintaining accurate measurement of transgene expression levels.
4Measurement precision
If gene-specific primers are used to differentiate transgenic expression, then measurement precision is improved, but ease of operation deteriorates
Solution Approach 1:
The method simplifies operation by using a universal primer set that automatically differentiates transgenic from endogenous expression through the presence of the heterologous terminator sequence. This eliminates the complex process of designing and validating multiple gene-specific primers for each transgene, making the assay easier to perform while maintaining precise differentiation capability.
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
This approach enables accurate and efficient quantification of transgene expression and copy number in transgenic plants, reducing costs and errors, and provides a method to distinguish transgenic from endogenous expression, facilitating high-throughput analysis.
Implementation Method 1
a forward primer and a reverse primer, wherein the forward primer and the reverse primer hybridize to the heterologous terminator sequence
Implementation Method 2
quantifying the level of expression of the heterologous polynucleotide by real-time reverse transcriptase polymerase chain reaction
Data Source
AI summary
The invention relates to a method for quantifying levels of expression and/or quantifying copy number of a heterologous polynucleotide in a transgenic plant using quantitative or real-time polymerase chain reaction (QPCR or real-time PCR), wherein the real-time PCR is performed using a primer set specific to a heterologous terminator sequence operably linked to the heterologous polynucleotide.