Nanopore Sequencing via Ionic Current Stochastic Sensing
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Solution Overview
Problem
Current DNA sequencing methods are costly, require sophisticated equipment, and are limited to sequences of less than one thousand nucleotides in length, with challenges in identifying nucleotides at the single molecular level, particularly in exonuclease sequencing where fluorescent labeling has limited success.
Innovation Solution
The method involves contacting nucleotides with a transmembrane protein pore to measure current amplitude, allowing for the identification and sequencing of nucleic acids through stochastic sensing, using a processive exonuclease to digest nucleotides and a cyclodextrin to facilitate interaction with the pore, enabling sequencing without expensive reagents like fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labeling is used to identify nucleotides in exonuclease sequencing, then nucleotide identification is enabled, but the method becomes costly and complex
Solution Approach 1:
The patent extracts the identification function from complex fluorescent labeling systems and relocates it to simple ionic current measurement through nanopores. By removing the need for fluorescent dyes and sophisticated detection equipment, the method achieves nucleotide identification through purely electrical signals generated by the nucleotides themselves interacting with the pore.
Solution Approach 2:
The patent replaces optical detection systems (fluorescent labeling and imaging) with electrical detection systems (ionic current measurement). This substitution eliminates the need for complex optical components, filters, and detectors, reducing overall system complexity while maintaining identification capability.
2Measurement precision
If conventional DNA sequencing methods are used, then sequencing accuracy is achieved, but the method is limited to sequences of less than one thousand nucleotides in length
Solution Approach 1:
The patent enables continuous sequencing of long DNA molecules by maintaining constant ionic current flow through the nanopore throughout the entire sequencing process. Unlike conventional methods that process segments separately, the continuous measurement capability allows uninterrupted sequencing of sequences exceeding one thousand nucleotides while maintaining accuracy.
Solution Approach 2:
The patent transitions from sequential, segment-based sequencing to simultaneous, continuous sequencing by measuring ionic current changes across the entire molecule passage through the pore. This dimensional shift from discrete segments to continuous measurement enables handling of much longer sequences.
3Reliability
If sophisticated equipment and reagents are used in conventional sequencing, then sequencing reliability is improved, but the cost and operational complexity increase
Solution Approach 1:
The patent replaces expensive, sophisticated sequencing equipment with simple, inexpensive nanopore structures and basic electrical measurement devices. The reliability is maintained not through complex machinery but through the robustness of the simple ionic current measurement principle and the stability of the nanopore-nucleotide interaction system.
Solution Approach 2:
The patent makes the sequencing system self-sufficient by using the nucleotides themselves as the signal generators through their inherent ionic current interaction with the pore, eliminating the need for external fluorescent labels, enzymes, or complex reagents. The system serves itself without requiring sophisticated supporting components.
4Ease of operation
If exonuclease sequencing is used to digest DNA one nucleotide at a time, then sequencing simplicity is improved, but the difficulty in identifying released nucleotides at single molecular level holds back development
Solution Approach 1:
The nanopore serves as an intermediary detection device that translates the presence and identity of individual released nucleotides into measurable ionic current changes. This intermediary function bridges the gap between the simple exonuclease digestion process and the detection requirement, enabling single-molecule nucleotide identification through electrical signals.
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 provides a rapid, simple, and cost-effective DNA sequencing method at the single molecule level, capable of sequencing beyond the length limitations of existing methods, with high accuracy in identifying individual nucleotides.
Implementation Method 1
stochastic sensing involves placing a nanometer sized pore in an insulating lipid bilayer membrane and measuring the ionic transport through the pore. When an analyte interacts with a binding site within the pore, a change in the ionic current is detected
Implementation Method 2
measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide
Implementation Method 3
digesting an individual nucleotide from one end of the target sequence using a processive exonuclease
Implementation Method 4
Various binding sites can be created within the pore by way of protein mutation, chemical modification, and by use of molecular adaptors and carriers
Data Source
AI summary
The invention relates to a method of identifying an individual nucleotide, comprising (a) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore and (b) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide. The invention also relates to a method of sequencing nucleic acid sequences and kits related thereto.