Nanopore Sequencing With Clock Nucleotides for Signal Discrimination
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Solution Overview
Problem
Current nucleic acid sequencing technologies are slow and expensive due to reliance on amplification techniques and require large volumes of specialist chemicals, while nanopore sequencing faces challenges in resolving overlapping k-mer signals and enzyme-assisted translocation inefficiencies.
Innovation Solution
The method involves expanding the target polynucleotide using clock and signal nucleotides with predetermined sequences to improve signal discrimination, allowing for reliable identification of individual nucleotides through a nanopore by deriving analysis signals from clock and nucleotide signals during translocation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification techniques are used to produce large volumes of nucleic acid, then sequencing sensitivity is improved, but sequencing speed decreases and cost increases
Solution Approach 1:
The patent extracts and eliminates the amplification step from the sequencing workflow by using nanopore technology that can directly sequence single molecules of nucleic acid. This removal of the amplification technique allows sequencing to proceed without the time-consuming and costly PCR processes, thereby improving sequencing speed while maintaining sensitivity through direct detection of individual molecules at the nanopore.
2Measurement precision
If amplification techniques are used to produce large volumes of nucleic acid, then sequencing sensitivity is improved, but reagent consumption increases
Solution Approach 1:
The patent removes the amplification step that consumes large amounts of specialist chemicals and reagents. By using nanopore sequencing on single molecules, the method eliminates the need for PCR reagents, fluorescent chemicals, and other consumables required in traditional sequencing, thereby reducing reagent consumption while maintaining sequencing sensitivity through direct electrical detection.
3Measurement precision
If enzyme-assisted translocation is used to control DNA movement through nanopore, then nucleotide resolution is improved, but translocation efficiency decreases due to missed states
Solution Approach 1:
The patent changes the translocation mechanism from enzyme-assisted to voltage-driven direct translocation. By applying an electric field across the nanopore, DNA molecules are driven through the pore based on their charge, eliminating the need for enzymatic control. This parameter change from biochemical to physical control improves translocation efficiency by avoiding missed states while maintaining nucleotide resolution through the inherent sensitivity of the nanopore electrical measurements.
4Measurement precision
If k-mer based measurement is used in nanopore sequencing, then signal discrimination is improved, but signal overlap occurs reducing measurement accuracy
Solution Approach 1:
The patent segments the k-mer measurement approach into individual nucleotide measurements by using a nanopore configuration that resolves single nucleotides. Instead of measuring signals from multiple nucleotides simultaneously (k-mers), the method segments the measurement process to detect each nucleotide separately as it passes through the nanopore, thereby eliminating signal overlap and improving measurement accuracy while maintaining signal discrimination capability.
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 enhances the reliability of nucleotide identification and reduces the number of contributing nucleotides per measurement, enabling faster and cheaper sequencing by improving signal discrimination and overcoming nanopore limitations.
Implementation Method 1
measuring voltage-driven ion flow through the nanopore in the presence of nucleotides of the polynucleotide
Implementation Method 2
measuring voltage-driven ion flow through the nanopore
Implementation Method 3
information about the nucleotides may be revealed by distinctive ion current signatures, such as the duration and extent of current block and the variance of current levels
Data Source
AI summary
A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.