Nucleic Acid Sequencing via Solid-Phase Immobilization and Optical Detection
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges with high reagent consumption and time requirements, limited read lengths, and accuracy issues, making them inefficient for routine human genome sequencing.
Innovation Solution
A method involving the fixation of nucleic acids in a stretched form on a substrate, followed by denaturation and interaction with oligonucleotide probes to form heteroduplexes, allowing for precise optical detection and sequencing without the need for extensive amplification or reagent exchange.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If gel electrophoresis-based sequencing methods are used, then sequencing accuracy is achieved, but time consumption and cost increase significantly
Solution Approach 1:
The patent extracts and eliminates the gel electrophoresis step from the sequencing process. By using solid-phase reversible immobilization of nucleic acids on a chip surface, the method removes the time-consuming gel electrophoresis separation step while maintaining sequencing capability through direct optical detection of fluorescently labeled nucleotides incorporated at the immobilized template.
Solution Approach 2:
The patent replaces the mechanical gel electrophoresis system with an optical detection system. Instead of using electrical fields and gel matrices to separate and detect nucleotides, the invention uses fluorescent labels and optical imaging to detect incorporated nucleotides directly at the immobilized template location, dramatically reducing time while maintaining accuracy.
2Productivity
If Illumina sequencing with reversible terminators is used, then large-scale sequencing is achieved, but read length is limited and reagent consumption increases
Solution Approach 1:
The patent performs preliminary immobilization of the nucleic acid template on the chip surface before sequencing begins. This preliminary action allows the template to be fixed in place and ready for sequential nucleotide incorporation without requiring repeated sample handling or reagent exchange, enabling longer reads and reducing reagent consumption while maintaining high throughput.
3Length of stationary object
If Pacific Biosciences SMRT sequencing is used, then long read lengths are achieved, but throughput is reduced due to fixed detector positioning
Solution Approach 1:
The patent transitions from a single fixed detector position to a two-dimensional array of detection sites on a chip surface. By immobilizing multiple templates across a spatial array and using corresponding spatially distributed detectors, the system achieves both long read lengths (through continuous observation like PacBio) and high throughput (through parallel observation of multiple templates simultaneously), effectively adding a spatial dimension to the detection architecture.
4Length of stationary object
If Oxford Nanopores sequencing is used, then very long read lengths are achieved, but accuracy decreases significantly
Solution Approach 1:
The patent introduces fluorescently labeled nucleotides as intermediaries in the sequencing process. Instead of directly detecting the nucleic acid passing through a nanopore, the method uses fluorescent labels on incorporated nucleotides as mediators that emit detectable signals when excited. This intermediary approach enables long-read sequencing with high accuracy by providing clear optical signals for base identification while maintaining the continuous observation capability needed for long reads.
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, long-range haplotype-resolved sequencing with improved accuracy and reduced reagent use, addressing the limitations of existing methods by enabling precise localization and sequencing of nucleic acids with high coverage and precision.
Implementation Method 1
exposing the fixed first strand and the fixed second strand to a respective pool of a respective oligonucleotide probe in a set of oligonucleotide probes, where each oligonucleotide probe in the set of oligonucleotide probes is of a predetermined sequence and length. The exposing (c) occurs under conditions that allow for individual probes of the respective pool of the respective oligonucleotide probe to bind and form a respective heteroduplex with each portion of the fixed first strand or the fixed second strand that is complementary to the respective oligonucleotide probe
Implementation Method 2
giving rise to a respective instance of optical activity
Data Source
AI summary
Systems and methods for determining a sequence of at least a portion of a target polymer from a subject are provided. A dataset that comprises one or more image files is obtained. A combined plurality of localizations based at least in part on each respective plurality of fluorophore localizations is determined for each image file in the one or more image files. Each localization in the combined plurality of localizations includes a target polymer position identity and a spatial location. The plurality of localizations are segmented into one or more target polymer strands. Each target polymer strand corresponds to a respective subset of localizations and target polymer position identities. A respective target polymer sequence is assembled using each subset of localizations for each target polymer strand, thereby providing a set of target polymer sequences.