Substrate Nucleic Acid Amplification With Inhibitor Washout
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Solution Overview
Problem
Existing methods for nucleic acid preparation and analysis are time-consuming and complex, requiring multiple steps such as washing and elution, and are not compatible with subsequent process steps, especially when dealing with limited biological samples.
Innovation Solution
A method involving a substrate that allows simultaneous separation and amplification of nucleic acids by applying a sample to a sample application zone, followed by an aqueous buffer wash to dilute inhibitors, and then an isothermal nucleic acid amplification reaction mixture, where target nucleic acids with a first molecular weight are immobilized and amplification products with a second molecular weight migrate via lateral flow along the substrate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple washing and elution steps are used to separate nucleic acids, then purification quality is improved, but process complexity and time consumption increase
Solution Approach 1:
The patent combines multiple separate operations (washing, elution, separation, and amplification) into a single integrated substrate. The substrate performs washing by allowing buffer flow through its structure, elutes nucleic acids through controlled release mechanisms, and enables subsequent amplification reactions, thereby reducing process complexity while maintaining purification quality.
Solution Approach 2:
The substrate is designed to perform multiple functions simultaneously: it acts as a washing medium, an elution surface, a separation matrix, and an amplification platform. This multi-functionality eliminates the need for multiple separate substrates or processing steps, directly addressing the contradiction between purification quality and process complexity.
2Manufacturing precision
If multiple washing steps are performed, then nucleic acid purity is improved, but time consumption increases
Solution Approach 1:
Multiple washing and elution steps are merged into a single substrate-based process. The substrate's structure allows buffers to flow through and perform washing functions, while simultaneously enabling elution through controlled release, thereby achieving high purity without the time cost of multiple sequential steps.
3Reliability
If washing buffer is applied to remove inhibitors, then amplification reliability is improved, but process complexity increases
Solution Approach 1:
The substrate serves as a universal platform that combines washing (to remove inhibitors) and amplification functions. The same substrate that performs the washing step also enables subsequent amplification reactions, eliminating the need for separate processing steps and maintaining amplification reliability while reducing complexity.
4Reliability
If separate containers are used for amplification, then reaction control is improved, but device complexity increases
Solution Approach 1:
The patent merges the amplification reaction container with the washing/separation substrate. The substrate itself serves as the reaction platform, allowing amplification reagents to be applied directly to the same surface where washing and separation occur, thereby maintaining reaction control while simplifying the overall device structure.
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 method simplifies the process by combining separation and amplification steps, enabling efficient nucleic acid analysis even with limited samples, without the need for external forces or specialized skills, and allows for on-site isolation.
Implementation Method 1
one or more inhibitors present on the sample application zone; applying an aqueous buffer to the sample application zone to wash away or dilute one or more inhibitors present on the sample application zone
Implementation Method 2
the target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and the amplification product has a second molecular weight lower than about 50kb migrates via lateral flow along the length of the substrate
Data Source
Figure 1~2
Figure 3~4
Figure 5A~5B
AI summary
A method is provided herein, the method includes: applying a sample comprising target nucleic acids to a sample application zone of a substrate; applying an aqueous buffer to the sample application zone of the substrate to washes away one or more inhibitors present on the sample application zone; and applying an isothermal nucleic acid amplification reaction mixture to the sample application zone to amplify the target nucleic acid to form a nucleic acid amplification product. The target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and wherein the amplification product having a second molecular weight.