Two-Phase PCR Process for Rapid Nucleic Acid Detection
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Solution Overview
Problem
Current PCR technologies are limited by lengthy processing times, which hinder fast and efficient nucleic acid amplification, particularly in applications requiring rapid diagnostics like infectious disease detection, where reducing the time and increasing throughput is crucial.
Innovation Solution
A two-phase PCR process is introduced, where primers with high melting temperatures are used in the first phase to accelerate primer extension, and probes with lower melting temperatures anneal and emit fluorescence signals in the second phase, optimizing thermal cycles and reducing overall processing time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If a smaller volume of PCR reaction mixture is used to reduce processing time, then the PCR process speed is improved, but the probability to amplify or detect target sequences at very low concentrations deteriorates
Solution Approach 1:
The PCR process is divided into two distinct phases: a first phase with higher annealing temperature (50-65°C) for initial amplification, and a second phase with lower annealing temperature (30-50°C) for enhanced detection sensitivity. This segmentation allows the reaction to benefit from both faster initial cycling and improved low-concentration target detection in the second phase, resolving the contradiction between speed and detection reliability.
2Reliability
If standard PCR thermal cycling is used to ensure specific amplification, then amplification specificity is maintained, but the processing time increases
Solution Approach 1:
The annealing temperature is dynamically adjusted between two phases: the first phase uses a higher temperature (50-65°C) to maintain specificity during initial amplification, while the second phase transitions to a lower temperature (30-50°C) to enhance detection sensitivity. This dynamic temperature adjustment allows the system to optimize both specificity and sensitivity without requiring extended processing time at a single suboptimal temperature.
3Productivity
If high-speed DNA polymerase is used to reduce PCR time, then processing speed is improved, but enzyme activity becomes affected by enzyme and template concentrations
Solution Approach 1:
The amplification process is segmented into two phases with different temperature conditions. The first phase uses higher annealing temperature (50-65°C) where standard polymerase activity is sufficient for rapid initial amplification. The second phase uses lower annealing temperature (30-50°C) where enzyme activity is more stable and less sensitive to concentration variations, allowing reliable detection of low-concentration targets without requiring specialized high-speed polymerases.
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 significantly shortens the PCR process, enhancing the speed and specificity of nucleic acid amplification, allowing for faster detection and quantification of target DNA, even at low concentrations, while minimizing nonspecific binding and primer-dimer formation.
Implementation Method 1
the reaction mixture includes at least one probe configured to emit a fluorescence signal upon excitation
Implementation Method 2
the primers anneal or bind to each DNA strand, creating a starting point for the addition of nucleotides
Implementation Method 3
the processing includes heating the reaction mixture to a first temperature and cooling the reaction mixture to a second temperature repeatedly for a first plurality of cycles
Data Source
AI summary
Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.