Gap-Probe Nucleic Acid Sequencing for Single-Molecule Phenotyping
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Solution Overview
Problem
Current single-molecule sequencing methods fail to link the functional properties of a DNA molecule (single-molecule phenotype) with its sequence, requiring specialist equipment and lacking high-throughput capabilities.
Innovation Solution
A method involving immobilizing nucleic acids on a surface, forming a single-stranded gap section, and using fluorescently labeled oligonucleotide probes to sequence and identify nucleotides by detecting their binding to the gap section, allowing for sequencing and phenotyping at the single-molecule level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-molecule sequencing methods are used, then the ability to sequence individual DNA molecules is achieved, but the capability to link functional properties with DNA sequence is lost
Solution Approach 1:
The patent introduces an intermediary system consisting of surface-immobilized nucleic acid molecules with attached functional elements (such as proteins or other biomolecules). This intermediary structure allows simultaneous observation of functional interactions and sequencing of the immobilized nucleic acid, thereby bridging the gap between functional properties and genetic sequence information.
2Measurement precision
If specialist equipment is used for single-molecule sequencing, then sequencing capability is achieved, but high-throughput capability and ease of operation are reduced
Solution Approach 1:
The patent segments the sequencing process into multiple independent steps that can be performed on standard equipment. The nucleic acid is divided into regions with distinct functions: immobilization regions, functional element attachment regions, and sequencing regions. This segmentation allows parallel processing of multiple samples and eliminates the need for specialized single-molecule sequencing instruments.
Solution Approach 2:
The patent employs universal adapters and primers that can be used across different nucleic acid samples and functional elements. The surface immobilization protocol and sequencing methodology are designed to be broadly applicable to various DNA and RNA molecules, enabling high-throughput processing using conventional laboratory equipment rather than specialized instruments.
3Measurement precision
If nucleic acid is immobilized on a surface for sequencing, then sequencing and phenotyping at single-molecule level is enabled, but the complexity of the procedure increases
Solution Approach 1:
The patent performs preliminary actions by pre-immobilizing the nucleic acid molecules on the surface and attaching functional elements before the sequencing step. The nucleic acid is prepared with specific structural features (such as hairpin structures or adapter sequences) that facilitate subsequent sequencing reactions. This preliminary preparation simplifies the actual sequencing process and reduces procedural complexity during data collection.
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
Enables sequencing and linking functional effects to nucleic acid sequences, facilitating the identification of sequence-dependent processes and interactions with biomolecules, and providing high-throughput capabilities.
Implementation Method 1
forming a single-stranded gap section by partially duplexing the nucleic acid such that the single-stranded gap section is flanked by duplex sections
Implementation Method 2
providing a set of at least four fluorescently labelled oligonucleotide probes... detecting binding, or absence thereof, of the fluorescently labelled oligonucleotide probes with the gap section
Data Source
Figure 1A~1D
Figure 2A~2D
Figure 3A~3D
AI summary
This invention relates to a method of sequencing a nucleic acid molecule, in particular the method comprising: (a) providing the nucleic acid immobilised on a surface; (b) forming a single-stranded gap section by partially duplexing the nucleic acid such that the single-stranded gap section is flanked by duplex sections, wherein the sequence to be sequenced is a sequence of the single-stranded gap section; (c) providing a set of at least four fluorescently labelled oligonucleotide probes, (d) detecting binding, or absence thereof, of the fluorescently labelled oligonucleotide probes with the gap section of the immobilised nucleic acid, wherein the identity of the interrogated nucleotide of the gap section of the immobilised nucleic acid is identified as the complementary base of the nucleotide X of the fluorescently labelled oligonucleotide probe that has the highest incidence of binding; (e) repeating steps (c) and (d) for interrogating subsequent nucleotide positions of the gap section of the immobilised nucleic acid until sufficient nucleotides of the gap section have been identified to be able to determine a sequence. Also provided are methods of single-molecule phenotyping and sequencing; methods of single-molecule phenotyping and identification of a molecule tagged with nucleic acid; an immobilised nucleic acid molecule; and a composition comprising oligonucleotide probes.