Extreme RT-PCR Using Trehalose and High Enzyme Concentrations
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current PCR instruments perform poorly with rapid denaturation and annealing times, leading to limited reproducibility and high variability due to thermal delays and temperature variance, which hinders the achievement of fast and efficient DNA amplification, especially in extreme PCR protocols requiring cycle times under 20 seconds.
Innovation Solution
The use of high concentrations of primers and polymerase, along with a sugar like trehalose, in combination with extreme temperature cycling profiles that allow for cycle times under 20 seconds, ensures robust PCR performance and yield by optimizing denaturation, annealing, and extension processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If conventional PCR instruments are used with standard cycling protocols, then thermal stability and reproducibility are maintained, but cycle time cannot be reduced below 20 seconds due to thermal delays and temperature variance
Solution Approach 1:
The patent applies parameter changes by optimizing primer concentration (increasing to 2-20 μM), polymerase concentration (increasing to 0.5-10 μM), and adding trehalose (0.1-1 M) to the reaction mixture. These parameter changes enable the reaction to tolerate extreme temperature cycling rates (≥100°C/s) while maintaining amplification efficiency and specificity, thus achieving cycle times under 20 seconds without sacrificing reproducibility
Solution Approach 2:
The patent uses beforehand cushioning by incorporating trehalose into the PCR reaction mixture before cycling begins. Trehalose acts as a protective agent that stabilizes the reaction components during extreme temperature transitions, cushioning against the thermal stress and variability that would otherwise compromise reproducibility at ultra-fast cycling rates
2Productivity
If rapid temperature cycling is implemented to reduce cycle time, then productivity increases, but thermal delays and temperature variance cause poor performance and high variability
Solution Approach 1:
The patent changes reaction parameters by using high primer concentrations (2-20 μM), high polymerase concentrations (0.5-10 μM), and adding trehalose (0.1-1 M). These parameter changes create a reaction system that is robust to extreme temperature cycling, enabling rapid amplification (cycle times <20 seconds) while maintaining consistent and reproducible results across replicates
Solution Approach 2:
The patent employs disposable optical reaction tubes or plates with thin walls designed for rapid heat transfer. These disposable components enable extreme temperature cycling rates by minimizing thermal mass and maximizing thermal conductivity, allowing rapid heating and cooling while maintaining amplification consistency across multiple use
3Speed
If extreme temperature cycling profiles with cycle times under 20 seconds are used, then reverse transcription speed increases, but conventional instruments cannot achieve the required heating and cooling rates
Solution Approach 1:
The patent changes reaction parameters (high primer concentration, high polymerase concentration, addition of trehalose) to create a system that can withstand and benefit from extreme temperature cycling rates. This enables reverse transcription and amplification to proceed efficiently even when instruments cycle temperatures at ≥100°C/s, achieving rapid RT-PCR without requiring complex custom-built instrumentation
4Reliability
If standard primer and polymerase concentrations are used, then reaction robustness is maintained under conventional cycling, but amplification efficiency drops at extreme cycling rates
Solution Approach 1:
The patent applies parameter changes by increasing primer concentration to 2-20 μM, increasing polymerase concentration to 0.5-10 μM, and adding trehalose at 0.1-1 M. These parameter changes create a reaction system that maintains high robustness and reliability while achieving excellent amplification efficiency and yield under extreme temperature cycling conditions with cycle times under 20 seconds
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 efficient and specific DNA amplification with high yields even at extreme cycle times, retaining reaction robustness and allowing for faster reverse transcription in RT-PCR, effectively overcoming the limitations of conventional PCR instruments.
Implementation Method 1
reverse transcribing the RNA to DNA by incubating for no longer than 5 minutes
Implementation Method 2
amplifying the DNA by polymerase chain reaction by thermally cycling the biological sample
Implementation Method 3
thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature
Implementation Method 4
primers configured for amplification of the target RNA to the biological sample
Data Source
Figure 1a
Figure 1b
Figure 1c
AI summary
Methods, kits and mixtures are provided for performing RT-PCR with an RT incubation of no more than one minute and PCR cycles in <20 seconds per cycle.