Nucleic Acid Release Agent for Direct RNA PCR Amplification
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Solution Overview
Problem
Current PCR methods for amplifying RNA samples are complex and prone to contamination due to the instability and degradation of single-stranded RNA, requiring stringent pretreatment and purification processes, which can lead to reduced sensitivity and accuracy in detection.
Innovation Solution
A nucleic acid release agent comprising Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycol, and a strong base, which allows for direct RNA release at room temperature, protecting nucleic acids and reverse transcriptase from alkaline environments, enabling direct PCR amplification without the need for extraction and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR methods are used for RNA amplification, then detection can be performed, but the process requires complicated pretreatment and purification steps due to RNA instability
Solution Approach 1:
The patent combines cell lysis, RNA release, and protection from degradation into a single buffer solution containing specific concentrations of Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycol, and a strong base. This merged solution eliminates the need for separate pretreatment and purification steps while maintaining RNA stability and detection reliability.
Solution Approach 2:
The patent introduces a specially formulated buffer solution as an intermediary that mediates between the RNA sample and the PCR reaction. This buffer solution protects RNA from degradation during the amplification process while allowing direct PCR amplification, thus simplifying the overall process without compromising detection stability.
2Measurement precision
If RNA samples undergo stringent pretreatment and purification, then detection accuracy improves, but the process time increases
Solution Approach 1:
The patent performs preliminary protection of RNA against degradation by incorporating specific buffer components (Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycol, and strong base) that create a protective environment before the PCR reaction begins. This preliminary action ensures RNA stability throughout the amplification process without requiring time-consuming purification steps.
3Productivity
If heating is used for sample treatment, then nucleic acid release is achieved, but aerosol contamination occurs
Solution Approach 1:
The patent changes the parameters of the lysis process by using a chemically optimized buffer solution that enables effective cell lysis and RNA release at room temperature or lower temperatures, eliminating the need for heating. The specific combination of Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycol, and strong base creates conditions that achieve efficient nucleic acid release without generating aerosol contamination.
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 solution simplifies RNA amplification and detection, achieving high sensitivity and repeatability while preventing RNA degradation and contamination, allowing for one-chamber, pollution-free, rapid PCR amplification and detection.
Implementation Method 1
a certain proportion of the strong base lyses cells, which release nucleic acids
Implementation Method 2
Triton X-100, on the one hand, can protect nucleic acids, especially single-stranded RNA, allowing RNA to be stored in an alkaline environment
Implementation Method 3
Sodium chloride and potassium chloride protect nucleic acids by coordinating the balance of intracellular and extracellular ions
Implementation Method 4
Tween 20 and ethyl phenyl polyethylene glycol can protect reverse transcriptase, so that the reverse transcriptase works normally in the alkaline environment
Data Source
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AI summary
Disclosed are a nucleic acid release agent, a PCR amplification method and a PCR amplification kit. The nucleic acid release agent comprises Tris-HCl, sodium chloride, potassium chloride, tween 20, Triton X-100, ethyl phenyl polyethylene glycol and a strong base, the molar concentration of Tris-HCl being 0.5 mM to 500 mM, the molar concentration of sodium chloride being 20 mM to 500 mM, the volume percentage of Tween 20 being 0.1% to 2%, the volume percentage of Triton X-100 being 0.1% to 3%, the volume percentage of ethyl phenyl polyethylene glycol being 0.1% to 3%, the mass concentration of potassium chloride being 5 mg/mL to 8 mg/mL and the mass concentration of the strong base being 2 mg/mL to 50 mg/mL. The nucleic acid release agent can enable a sample containing RNA to release RNA directly at room temperature, and to be directly mixed with a PCR reaction solution for PCR amplification.