STR PCR Cycle Control Using qPCR DNA Quantitation
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Solution Overview
Problem
Current PCR protocols face challenges in accurately normalizing DNA concentrations across multiple samples, particularly in high-throughput, low-cost forensic STR analysis, due to imperfections in amplification efficiency and the need for sophisticated instrumentation, which is not justified in such applications.
Innovation Solution
A method and system for DNA sample preparation involving parallel qPCR and STR PCR in separate or shared chambers of a cartridge, where qPCR provides concentration information to determine the number of STR PCR cycles needed, allowing for concurrent or staggered amplification without initial normalization or volume changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional qPCR quantification followed by dilution is used to normalize DNA concentration, then accurate concentration control is achieved, but processing time increases and sophisticated instrumentation is required
Solution Approach 1:
The system performs preliminary quantification of DNA concentration through qPCR before the main STR amplification process. This preliminary measurement enables determination of the optimal number of amplification cycles needed, eliminating the need for time-consuming post-quantification dilution steps and allowing direct progression to targeted amplification.
Solution Approach 2:
The system changes the parameter being controlled from fixed dilution volume to variable amplification cycle number. By using the qPCR quantification data to determine the precise number of cycles needed for STR amplification, the system achieves concentration normalization through cycle adjustment rather than physical dilution, reducing processing time while maintaining accuracy.
2Measurement precision
If sophisticated instrumentation with low detection limits is used for accurate DNA quantification, then high accuracy concentration estimates are achieved, but device complexity and cost increase
Solution Approach 1:
The system uses a multi-functional approach where the same PCR instrument performs both qPCR quantification and STR amplification. By integrating these two functions into a single system, the patent eliminates the need for separate sophisticated quantification instrumentation, reducing device complexity and cost while maintaining accurate concentration measurement capabilities.
Solution Approach 2:
The system uses its own qPCR quantification capability to self-determine the optimal amplification parameters for subsequent STR PCR. This self-service approach eliminates the need for external sophisticated instrumentation, as the system uses its measured concentration data to automatically adjust its own amplification process.
3Productivity
If fixed number of amplification cycles is used for all samples, then processing efficiency is maintained, but concentration normalization accuracy deteriorates
Solution Approach 1:
The system transitions from a static fixed-cycle amplification approach to a dynamic cycle-number approach. By using qPCR quantification results to determine the specific number of amplification cycles needed for each sample based on its DNA concentration, the system dynamically adjusts processing parameters to achieve accurate normalization while maintaining overall processing efficiency.
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
Achieves accurate DNA concentration normalization without complex instrumentation, enabling efficient and rapid STR PCR processes suitable for forensic analysis, meeting quality standards while reducing processing time and cost.
Implementation Method 1
detecting a fluorescent signal produced during the qPCR
Implementation Method 2
Polymerase Chain Reaction ("PCR") is a technique used to create duplicates of selected sequences of DNA
Data Source
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Figure 1B
Figure 2A
AI summary
Performing sample quantitation and sample amplification may be performed in a sample cartridge or sample cartridges. Sample quantitation using qPCR may be performed during STR PCR on the sample. Samples need not be normalized prior to performing STR PCR. In certain embodiments, qPCR and STR PCR are performed on the same cartridge, optionally at the same time (or in real-time, or overlapping in time) and optionally using some or all of the same PCR apparatus. In other embodiments, qPCR and STR PCR are performed on different cartridges. Quantitation of the STR PCR sample may be performed without substantially delaying the STR PCR process.