Nucleic Acid Amplification with Reference Molecule for qPCR Accuracy
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Solution Overview
Problem
Commercially available real-time qPCR platforms face limitations due to instrument-to-instrument variability and insufficient quality control, particularly in handling PCR inhibitors, which affects the accuracy and reliability of gene expression quantification, making them unsuitable for widespread diagnostic use.
Innovation Solution
The method involves amplifying a target nucleic acid molecule in the presence of a detectable nucleic acid probe and a reference nucleic acid molecule with reduced or no secondary structure, allowing for accurate detection by minimizing the difference in yield signal between melting curves, and using curve fitting algorithms to scale the yield signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a detectable nucleic acid probe is used to amplify and detect target nucleic acid, then detection sensitivity is improved, but secondary structure formation in the probe binding site reduces amplification efficiency and detection accuracy
Solution Approach 1:
The patent applies preliminary action by designing and selecting reference nucleic acid molecules with reduced or no secondary structure in the probe binding site before the actual detection process. This pre-optimization of the reference molecule structure ensures that it will not form interfering secondary structures during amplification, thereby establishing reliable baseline measurements that correct for probe binding variations and improve both detection accuracy and amplification efficiency
Solution Approach 2:
The patent employs parameter changes by modifying the structural parameters of the reference nucleic acid molecule specifically at the probe binding site. By reducing or eliminating secondary structure formation through sequence selection or molecular design, the patent optimizes the physical-chemical parameters of the reference molecule to match ideal probe binding conditions, thereby resolving the contradiction between detection sensitivity and amplification efficiency
2Reliability
If replicate measurements are performed to control for false results, then quality control is improved, but sample consumption increases and workflow becomes more complicated
Solution Approach 1:
The patent introduces an intermediary element - a reference nucleic acid molecule with reduced secondary structure - that serves as an internal control within the same reaction mixture. This reference molecule allows for simultaneous normalization and quality control without requiring separate replicate measurements, thereby maintaining reliability while simplifying the workflow and reducing sample consumption
Solution Approach 2:
The patent merges the quality control function with the main detection assay by incorporating the reference nucleic acid molecule into the same amplification reaction. This combination allows replicate-level quality control to be achieved within a single reaction workflow, eliminating the need for separate replicate measurements and reducing both workflow complexity and sample consumption
3Measurement precision
If more assays are performed per test to compensate for low RNA yield, then detection sensitivity is maintained, but reagent consumption increases and cost increases
Solution Approach 1:
The patent applies self-service by having the reference nucleic acid molecule within the reaction mixture automatically normalize for variations in RNA yield and amplification efficiency. This internal normalization mechanism eliminates the need for additional compensatory assays, as the single reaction inherently provides sufficient detection sensitivity through the reference-corrected measurement of the target nucleic acid
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 enhances the accuracy of nucleic acid quantitation, reduces instrument variability, and improves the reliability of diagnostic results by minimizing the impact of secondary structures and PCR inhibitors, making the method more suitable for clinical diagnostics.
Implementation Method 1
a detectable nucleic acid probe that is capable of hybridizing to the target nucleic acid molecule
Implementation Method 2
amplifying the target nucleic acid molecule in the presence of a detectable nucleic acid probe
Implementation Method 3
determining the melting temperature of the detectable nucleic acid probe to the target nucleic acid molecule
Data Source
AI summary
The invention features compositions and methods that are useful for the measurement of the quantity of a nucleic acid target in a sample.