Bt11 DNA Quantification via 5' Junction Targeting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting and quantifying DNA from transgenic corn event Bt11 are limited in their ability to discriminate between different transgenic events and may not be sensitive enough for accurate low-level detection, especially in mixed samples, due to factors like primer selection and PCR protocol variability.
Innovation Solution
A method using specific primer pairs and probes designed to target the 5' junction sequence of Bt11 DNA, combined with quantitative real-time PCR, allows for the simultaneous detection and quantification of Bt11 DNA relative to an endogenous maize adh1 gene, providing improved sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional PCR methods using common genetic elements (promoters, terminators, marker genes) are used for detection, then the methodology is simple and uniform, but the method cannot discriminate between different transgenic events that differ only in coding sequence
Solution Approach 1:
The patent applies local quality by designing primers and probes that specifically target the unique 5' junction region where the transgene integrates into the host genome. This localized targeting allows discrimination between different transgenic events by detecting event-specific sequences at the integration site, while the rest of the detection methodology remains conventional and uniform.
Solution Approach 2:
The patent segments the detection approach by separating the detection of common genetic elements from the detection of event-specific junction sequences. By using specific primers that target only the 5' junction region, the method isolates the discrimination function to a specific genomic location, enabling event-specific detection while maintaining overall methodological simplicity.
2Measurement precision
If event-specific PCR methods are developed to distinguish between transgenic events, then the ability to discriminate between events is improved, but factors such as primer selection, PCR protocol variability, and initial DNA concentration influence limit applicability and sensitivity
Solution Approach 1:
The patent implements feedback through the use of fluorescently labeled probes that provide real-time monitoring of PCR amplification. The probe hybridizes specifically to the 5' junction sequence, and fluorescence signal intensity provides feedback on the presence and quantity of target DNA, enabling accurate quantification and reducing the influence of PCR protocol variability on results.
Solution Approach 2:
The patent uses a fluorescent probe as an intermediary between the target DNA and the detection system. The probe specifically binds to the 5' junction sequence and transfers energy to the fluorescent dye, providing a reliable and reproducible signal that is independent of initial DNA concentration variations and PCR protocol differences.
3Measurement precision
If quantitative real-time PCR with specific primers and probes targeting the 5' junction sequence is used, then the sensitivity and specificity for detecting Bt11 DNA are improved, but the device complexity and reagent requirements increase
Solution Approach 1:
The patent replaces traditional end-point detection methods with real-time fluorescent detection during the PCR amplification process. This substitution allows for quantitative measurement of DNA amplification in real-time, significantly improving detection sensitivity and specificity while using standard real-time PCR instrumentation that is increasingly common in laboratories.
Solution Approach 2:
The patent changes the detection parameter from endpoint fluorescence or gel electrophoresis to real-time fluorescent monitoring during amplification. By monitoring the fluorescence signal throughout the PCR process, the method achieves higher sensitivity and specificity for detecting low levels of Bt11 DNA while using readily available real-time PCR systems.
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 achieves a limit of detection of less than 0.04% and a limit of quantification of less than 0.08% Bt11 DNA concentration with high accuracy and precision, demonstrating improved sensitivity and reliability across various samples and laboratories.
Implementation Method 1
a fluorescent dye labeled probe comprising SEQ ID NO: 3, wherein the first pair of primers, when used in a nucleic-acid amplification reaction with genomic DNA from event Bt11 corn, produce a first amplicon comprising SEQ ID NO: 4
Implementation Method 2
thermal amplification (polymerase chain reaction (PCR)) using polynucleotide primers or DNA hybridization using nucleic acid probes
Data Source
AI summary
Improved methods of quantifying nucleic acids that are unique to a transgenic corn event designated Bt11 in a biological sample and compositions thereof are disclosed. The invention further relates to primer pairs used in the method that are unique to event Bt11.