Nanopore Sequencing via Ternary Complexes
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Solution Overview
Problem
Current DNA sequencing methods are time-consuming and expensive, and struggle with accurately positioning repetitive sequences and determining the length of tandem short repeats, which are crucial for personalized medicine, and existing nanopore technologies face challenges with single-stranded DNA due to self-hybridization and folding issues.
Innovation Solution
The method involves forming local ternary complexes along double-stranded biopolymer molecules using sequence-specific probes and detecting their positional information using a nanopore system, allowing for accurate sequencing by generating probe maps that provide relative or absolute positional information of the probes along the target molecule.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-stranded DNA is used in nanopore sequencing, then the nanopore can detect individual bases, but the DNA undergoes self-hybridization and folding which reduces reliability
Solution Approach 1:
The patent changes the physical state parameter of the DNA from single-stranded to double-stranded form. By using double-stranded DNA as the template and designing probes that bind to specific sequences, the method eliminates self-hybridization issues while maintaining detection capability through probe-DNA hybridization signals detected by the nanopore
Solution Approach 2:
The patent introduces sequence-specific probes as intermediary molecules that mediate between the DNA template and the nanopore detector. These probes bind to the double-stranded DNA and form detectable complexes, allowing indirect detection of DNA sequences while avoiding the problems of direct single-stranded DNA detection
2Loss of information
If standard DNA probes are used in Sequencing-By-Hybridization, then the method can determine subsequences, but large probe arrays are required and positional information is lost reducing productivity
Solution Approach 1:
The patent adds the dimension of positional information to the hybridization data. By detecting not just which probes bind but where they bind along the DNA molecule, the method transforms 2D hybridization patterns into 1D positional maps, enabling more efficient sequence reconstruction with fewer probes
Solution Approach 2:
The patent applies different detection strategies to different regions of the DNA molecule. By focusing detection on probe binding positions and using variable-range alignment algorithms that adapt to local sequence characteristics, the method improves sequencing efficiency without requiring uniform coverage across the entire genome
3Quantity of substance
If repetitive sequences are sequenced using known methods, then coverage is obtained, but accurate positioning is impossible reducing measurement precision
Solution Approach 1:
The patent uses feedback from multiple probe binding positions to resolve repetitive sequence ambiguity. By detecting the precise positions of multiple probes along the DNA and using variable-range alignment algorithms that incorporate positional feedback, the method can distinguish between identical repetitive elements at different locations and accurately assemble the complete sequence
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 enables efficient and accurate sequencing of biopolymers, particularly double-stranded DNA, by reducing self-hybridization issues and improving probe affinity and selectivity, thereby enhancing sequencing speed and accuracy while reducing costs.
Implementation Method 1
nanopore detection systems... passing the double-stranded biopolymer target molecule through a nanopore and detecting an electrical signal indicative of one or more locations of the one or more local ternary complexes
Implementation Method 2
contacting the target molecule with a plurality of probes each having specificity for one or more recognition sites of the target molecule, thereby forming local ternary complexes
Data Source
Figure 1
Figure 2~4A
Figure 4B
AI summary
Methods for sequencing a biopolymer by forming local ternary complexes along the length of the double- stranded biopolymer target molecule using one or more probes and obtaining information about the location of the probe(s) using a detector. These methods offer particular advantage when implemented with nanopore (including micropore) detection systems.