Microfluidic DNA Quantification Using Human-Alu Probes
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Solution Overview
Problem
Current nucleic acid quantification methods in forensic DNA analysis face challenges such as allele peak height imbalance, allele drop-out, stutter, non-specific band creation, and contamination from non-human DNA, which can lead to difficulties in interpreting STR profiles, especially when DNA concentrations are not within a narrowly defined range.
Innovation Solution
The development of microfluidic methods and devices that combine a sample fluid with a binding agent comprising a signaling moiety in a microfluidic channel, allowing for rapid detection and quantification of target nucleic acid, even at low concentrations, and in the presence of contaminants, using a microfluidic device with integrated laser detection and molecular beacon probes for precise nucleic acid analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid quantification methods are used, then DNA concentration can be measured, but artifacts including allele peak height imbalance, allele drop-out, stutter, non-specific band creation, and pull-up peaks occur when DNA concentration is not within the narrow range
Solution Approach 1:
The patent performs nucleic acid quantification before STR amplification to determine the appropriate sample amount. By measuring DNA concentration in advance using real-time PCR or other quantification methods, the system can adjust the volume of template DNA added to the multiplex PCR reaction, ensuring the final concentration falls within the optimal range for reliable STR profiling.
Solution Approach 2:
The system uses quantification results to feedback-adjust the amount of template DNA used in subsequent STR amplification. Based on the measured DNA concentration and quality metrics, the system dynamically determines the optimal sample volume to load, creating a closed-loop control that adapts to variations in sample quality and prevents artifacts.
2Quantity of substance
If total DNA quantification is used in forensic samples, then total nucleic acid amount can be measured, but human-specific DNA amount cannot be determined, leading to inappropriate amplification when non-human DNA contamination is present
Solution Approach 1:
The patent employs species-specific quantification by using human-Alu probe sequences that selectively bind only to human DNA. This allows differentiation between human and non-human DNA in mixed samples, enabling precise measurement of the human-specific component even when total DNA includes contaminating bacterial, fungal, or animal DNA.
Solution Approach 2:
The system uses human-Alu probe sequences as an intermediary to specifically detect and quantify human DNA in the presence of non-human contaminants. The probe acts as a selective mediator that binds only to human genomic DNA, allowing accurate measurement of the target nucleic acid while ignoring contaminating DNA.
3Productivity
If rapid quantification is performed within 1 hour, then productivity is improved, but measurement precision may be compromised
Solution Approach 1:
The patent implements continuous real-time monitoring during the quantification process using real-time PCR or similar continuous detection methods. Rather than relying on endpoint measurements, the system continuously tracks nucleic acid amplification or binding signals, allowing accurate quantification to be achieved rapidly through continuous data collection and analysis throughout the reaction process.
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 accurate and rapid quantification of nucleic acids, including those at low concentrations, while minimizing artifacts and contamination effects, facilitating reliable forensic DNA analysis and automation of nucleic acid processing.
Implementation Method 1
combining in a microfluidic channel the sample fluid and a binding agent comprising a signaling moiety, wherein the binding agent becomes immobilized with respect to the target nucleic acid
Implementation Method 2
detecting the signaling moiety
Data Source
AI summary
Described herein are methods and devices for nucleic acid quantification and, in particular, to microfluidic methods and devices for nucleic acid quantification. In certain embodiments methods of quantifying a target nucleic acid without the need for amplification are provided. The methods involve, in some embodiments, allowing a binding agent to become immobilized with respect to the target nucleic acid. In some cases, the binding agent comprises a signaling moiety that can be used to quantify the amount of target nucleic acid. In another aspect, the quantification can be carried out rapidly. For example, in certain embodiments, the quantification can be completed within 5 minutes. In yet another aspect, samples containing a low amount of target nucleic acid can be quantified. For instance, in some cases, samples containing less than 100 nanograms per microliter may be quantified. Also described are devices and kits for performing such methods, or the like.