Nucleic Acid Detection via Parallel PCR Segmentation
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Solution Overview
Problem
Current methods for determining the expression level of specific nucleic acid molecules, such as miRNAs, at low concentrations in biological samples are unreliable due to statistical variations in preamplification steps, leading to inconsistent results at the lower detection limit.
Innovation Solution
A method involving multiple independent PCR amplifications of nucleic acid molecules from a biological sample, followed by mixing equal amounts for PCR-based analysis, which allows for the determination of expression levels by calculating the mean value of PCR results, thereby reducing variability and improving accuracy at low concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a preamplification step is performed before real-time PCR to detect low concentration nucleic acid molecules, then the sensitivity of detection is improved, but the reliability and precision of measurement deteriorate due to statistical variations in preamplification
Solution Approach 1:
The patent divides the preamplification process into multiple independent parallel reactions (e.g., three separate preamplification reactions) instead of performing a single preamplification. Each reaction processes an aliquot of the original nucleic acid sample independently, and the results are averaged to obtain the final measurement. This segmentation reduces the impact of statistical variations in any single preamplification reaction, thereby improving measurement reliability while maintaining detection sensitivity.
2Measurement precision
If multiple independent PCR amplifications are performed and results are averaged, then the accuracy and reproducibility improve, but the complexity of the detection method increases
Solution Approach 1:
The method segments the detection process into multiple independent PCR amplification reactions, each processing an aliquot of the sample. By performing parallel reactions and averaging the results, the patent improves measurement accuracy and reproducibility. The segmentation approach allows the complexity to be distributed across multiple simple, standardized PCR reactions rather than requiring a single complex procedure.
Solution Approach 2:
The patent combines multiple independent PCR amplification results through averaging to obtain a final measurement. This merging of results from parallel reactions enhances the overall measurement quality by reducing random variations, while each individual reaction remains a standard, well-understood PCR process.
3Reliability
If multiple independent PCR amplifications are performed followed by mixing and single PCR analysis, then the variability in preamplification is reduced, but the time and resource consumption increase
Solution Approach 1:
The patent segments the sample into multiple aliquots for parallel preamplification reactions, which can be performed simultaneously. This parallel processing approach reduces the total time compared to sequential reactions, while still benefiting from the reduced variability achieved through multiple independent amplifications. The segmented approach allows efficient use of resources and time.
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 precise and reliable detection of nucleic acid molecules at extremely low concentrations, improving reproducibility and accuracy of results by averaging out statistical variations in preamplification processes.
Implementation Method 1
performing an independent polymerase chain reaction (PCR) with each of the three or more aliquots in order to amplify the specific nucleic acid molecule
Data Source
AI summary
The present invention relates to methods for the detection of nucleic acid molecules at the lower detection limit.