Nanopore Sequencing Secondary Structure Control
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Solution Overview
Problem
Current polynucleotide sequencing technologies are slow and expensive due to reliance on amplification techniques and require high quantities of specialist fluorescent chemicals for signal detection.
Innovation Solution
A method involving a transmembrane pore with a molecular brake to control polynucleotide movement, accompanied by conditions on the other side of the pore to manage secondary structure formation, allowing for improved characterization of polynucleotides through controlled secondary structure formation and rehybridization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification techniques and fluorescent chemicals are used for polynucleotide sequencing, then signal detection capability is improved, but cost and complexity increase
Solution Approach 1:
The patent extracts the detection function from complex fluorescent chemical systems and transfers it to a simple nanopore-based electrical sensing system. The nanopore directly detects polynucleotide translocation and secondary structure formation through current changes, eliminating the need for fluorescent labels and complex optical detection systems.
Solution Approach 2:
The patent replaces the optical/chemical detection system with an electrical/mechanical sensing system. Instead of using fluorescent chemicals and optical detectors, the system uses a nanopore to detect mechanical translocation and structural changes of polynucleotides through electrical current measurements.
2Measurement precision
If secondary structure formation is allowed to occur naturally, then polynucleotide characterization is improved, but measurement variability increases
Solution Approach 1:
The patent applies preliminary action by pre-configuring the trans side environment with specific conditions (salt concentration, temperature, pH) that are optimized to control secondary structure formation. This pre-prepared environment ensures that polynucleotides consistently form the desired secondary structures after translocation, reducing variability in measurements.
Solution Approach 2:
The patent changes physical and chemical parameters on the trans side of the nanopore (such as ionic strength, temperature, and pH) to control the formation and stability of secondary structures. By optimizing these parameters, the system achieves consistent secondary structure formation, improving measurement reliability.
3Speed
If molecular brake is used to control polynucleotide movement through pore, then translocation control is improved, but system complexity increases
Solution Approach 1:
The patent employs a molecular brake that utilizes the polynucleotide's own properties (such as base stacking interactions and electrostatic repulsion) to control its translocation speed. The molecular brake automatically regulates movement without requiring external control mechanisms, maintaining simplicity while achieving precise speed control.
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
Enhances measurement accuracy and reduces variability in polynucleotide sequencing by controlling secondary structure formation, leading to improved signal consistency and reduced need for fluorescent chemicals.
Implementation Method 1
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time.
Implementation Method 2
the conditions on the other side of the pore are selected to control the formation of secondary structure by the target polynucleotide on the other side of the pore
Implementation Method 3
the hairpin loop is designed to control the ability of the two strands of the target polynucleotide to rehybridise on the other side of the pore
Data Source
AI summary
The invention relates to a new method of characterising a target polynucleotide using a pore. The method involves controlling the formation of secondary structure by the target polynucleotide after the polynucleotide has moved through the pore.