Nucleic Acid Amplification Tube With Barrier Insert
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Solution Overview
Problem
Current convective PCR methods face challenges with low amplification efficiency, reduced specificity, and inconsistencies in nucleic acid amplification due to complex liquid flow paths and temperature control issues, leading to non-specific amplification and inaccurate quantification.
Innovation Solution
A reaction tube with a physical barrier insert that controls the liquid circulation path, allowing reagents to move under and over the insert, combined with temperature control to ensure specific temperature zones for denaturation and annealing, improving the efficiency and specificity of the amplification process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If metal block temperature control is used for PCR, then temperature control is achieved, but heating and cooling time is too long (2-3 hours)
Solution Approach 1:
The reaction system is segmented into distinct functional regions: a heated region at the bottom for denaturation, a cooler region at the top for annealing, and an intermediate region for extension. The insert divides the reaction chamber into upper and lower spaces, creating spatial separation of temperature zones that enables simultaneous occurrence of different PCR steps without requiring global heating/cooling cycles.
Solution Approach 2:
Different regions of the reaction tube are given different thermal properties and temperature conditions. The bottom region is heated to high temperature for denaturation, while the top region remains cooler for annealing. This local differentiation of thermal conditions allows multiple PCR steps to occur concurrently in different spatial locations.
2Productivity
If Rayleigh-Benard convection is used for rapid PCR, then amplification speed is rapid, but reagent leakage and contamination occur
Solution Approach 1:
The insert acts as a physical barrier and intermediary structure that guides the convective flow of reagents. It prevents direct contact between the heated bottom region and the cooler top region, forcing reagents to flow through controlled pathways. This intermediary structure maintains the benefits of convection-driven rapid mixing while preventing leakage and contamination by containing reagents within defined flow channels.
3Object-affected harmful factors
If natural convection is used in open reaction tube, then leakage and contamination are solved, but flow path is complicated and amplification efficiency is low
Solution Approach 1:
The reaction tube is segmented by the insert into distinct upper and lower spaces with defined flow paths. This segmentation simplifies the otherwise complicated natural convection flow into more predictable and efficient circulation patterns, where reagents systematically pass through heated and cooled regions in a controlled manner, improving amplification efficiency.
4Productivity
If complex multilayer flow path is formed in convection PCR, then circulation occurs, but denaturation and annealing efficiency are low
Solution Approach 1:
The insert creates localized temperature zones with distinct thermal characteristics. The lower space near the heat source maintains high temperature for effective denaturation, while the upper space remains cooler for effective annealing. This local quality differentiation ensures that reagents experience appropriate temperature conditions in each region, improving the efficiency of denaturation and annealing reactions.
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 enhances denaturation and annealing efficiency, reduces non-specific amplification, and improves consistency among tubes, enabling real-time quantification and accurate amplification results.
Implementation Method 1
an insert is disposed in said nucleic acid amplification region with an upper space remained above the insert and a lower space remained below the insert. When a reagent is injected into the reaction tube, the reagent is capable of moving along a circulation path through the upper space and the lower space in the reaction tube under an internal force or external force, due to a physical barrier effect of the insert
Implementation Method 2
using two constant temperature heat sources disposed at upper and lower regions respectively to establish a closed reaction cavity which has a temperature gradient from bottom to top, and thus convective motion of the PCR reagents occurs spontaneously
Implementation Method 3
a method named Rayleigh-Benard PCR (RB-PCR), based on the principle of heat conduction and thermal convection
Implementation Method 4
based on the principle of heat conduction and thermal convection, using two constant temperature heat sources disposed at upper and lower regions respectively
Data Source
Figure 1a~1b
Figure 2a~2d
Figure 3
AI summary
Disclosed are a reaction tube for nucleic acid amplification capable of controlling a liquid circulation path, a reaction apparatus for nucleic acid amplification comprising the reaction tube, and a method for amplifying nucleic acid comprising a step of using the reaction tube. Also disclosed are a kit comprising the reaction tube, and use of the reaction tube in preparation of a kit.