Fluorescent Dye Thermocycling Feedback for PCR Accuracy
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Solution Overview
Problem
Current PCR technologies face challenges in accurately measuring and controlling the temperature of the PCR solution during thermal cycling, especially at faster speeds, due to inaccuracies in temperature measurement and control, which affects the consistency of DNA amplification.
Innovation Solution
The use of a temperature-sensitive fluorescent dye, such as sulforhodamine B, that emits a luminescent signal in response to temperature changes, allowing for real-time monitoring of the sample temperature and precise control of thermocycling through fluorescence-based feedback.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If external temperature sensors are used to measure the temperature of the PCR solution, then the measurement is non-intrusive and does not contaminate the sample, but the solution temperature frequently lags behind the instrument block or chamber temperature during temperature transitions, leading to inaccuracy and inconsistency
Solution Approach 1:
The patent replaces the mechanical/physical temperature sensor system with an optical fluorescence-based temperature sensing system. Temperature-sensitive fluorescent dyes are added to the PCR solution, and their fluorescence properties change with temperature. This optical method allows direct measurement of solution temperature without physical contact, eliminating the thermal lag inherent in physical sensors while maintaining non-intrusiveness.
Solution Approach 2:
The patent introduces temperature-sensitive fluorescent dyes as intermediaries between the PCR solution and the temperature measurement system. These dyes absorb light at one wavelength and emit fluorescence at another wavelength, with the fluorescence intensity or wavelength shifting in response to temperature changes. This intermediary mechanism enables indirect temperature measurement that accurately reflects solution temperature without the limitations of direct physical sensing.
2Measurement precision
If direct physical sensors are placed within the PCR solution, then accurate solution temperature measurement can be obtained, but product contamination, PCR inhibition, added thermal mass, and obstruction of optical measurements occur
Solution Approach 1:
The patent replaces physical contact-based temperature sensing with an optical fluorescence-based sensing system. Temperature-sensitive fluorescent dyes are dissolved in the PCR solution and respond to temperature changes through changes in their fluorescence properties. This eliminates the need for physical sensors that would contaminate the sample, inhibit PCR, add thermal mass, or obstruct optical measurements, while still providing accurate solution temperature measurement.
3Productivity
If PCR cycling speeds are increased to improve productivity, then more cycle time is spent in temperature transition, but the solution temperature seldom tracks the measured instrument temperature, making accurate temperature measurement the limiting factor
Solution Approach 1:
The patent replaces traditional physical temperature sensors with an optical fluorescence-based temperature sensing system using temperature-sensitive dyes. This substitution enables accurate real-time temperature measurement during rapid temperature transitions, as the fluorescent dyes respond quickly to temperature changes without the thermal inertia of physical sensors. This allows fast PCR cycling to proceed with maintained temperature measurement accuracy.
Solution Approach 2:
The patent implements feedback control by continuously monitoring the fluorescence signal from temperature-sensitive dyes and using this information to adjust the temperature cycling profile. The real-time temperature data obtained from fluorescence measurements provides feedback to the thermal cycler control system, enabling precise temperature control even during rapid cycling, thereby maintaining measurement accuracy as the limiting factor is overcome.
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 accurate and precise temperature control within the PCR solution, improving the consistency and efficiency of DNA amplification by directly measuring the internal temperature, reducing temperature discrepancies between the solution and the instrument, and enhancing the accuracy of thermocycling.
Implementation Method 1
The use of a temperature-sensitive fluorescent dye, such as sulforhodamine B, that emits a luminescent signal in response to temperature changes
Data Source
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AI summary
Systems, methods, and kits are provided wherein a temperature-sensitive reagent that emits a luminescent signal is used to adjust the identification of the temperature of a sample or to control thermocycling. In various illustrative embodiments, the sample is a PCR mixture.