Fluorescent Polynucleotide Sequencing With Light-Blocking Reaction Wells
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Solution Overview
Problem
Existing nucleic acid sequencing methods face challenges such as high cost, complexity, and inaccuracies due to photodamage, unstable reaction environments, and misidentification of nucleotides, necessitating the development of a more efficient and accurate sequencing technique.
Innovation Solution
A fluorescence-based method using a solid state substrate with an opaque metal layer and a reaction well that blocks excitation light, where a fluorescently labeled polynucleotide strand is cleaved by an exonuclease to release mononucleotides, allowing for accurate sequencing through detection of fluorescent signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescently labeled polynucleotide strands are used for sequencing, then sequencing accuracy is improved, but photodamage occurs that inactivates polymerases and destroys labels
Solution Approach 1:
The patent extracts the harmful excitation light from the reaction environment by using an opaque substrate that blocks light, thereby removing the cause of photodamage while preserving the fluorescent labeling method for accurate sequencing
Solution Approach 2:
The patent converts the harmful effect of excitation light into a beneficial detection mechanism by using fluorescent labels that emit light only when excited, allowing accurate nucleotide identification while minimizing overall light exposure to prevent photodamage
2Productivity
If next generation sequencing methods are used, then throughput is improved, but sequence gaps and ambiguities increase due to alignment of short sequences
Solution Approach 1:
The patent segments the sequencing process into discrete, controllable steps using solid-state reaction wells with precise geometric constraints, enabling systematic analysis of each segment without the alignment complexities of whole-genome approaches
Solution Approach 2:
The patent performs preliminary actions by pre-positioning fluorescently labeled polynucleotide strands in reaction wells before excitation, ensuring that sequencing occurs in a controlled manner that prevents gaps and ambiguities from the outset
3Adaptability or versatility
If protein nanopores are used for sequencing, then sequencing capability is achieved, but hydrophobic environments are required that are unstable and difficult to manufacture
Solution Approach 1:
The patent replaces the complex protein nanopore system with a simple solid-state substrate containing geometric openings, eliminating the need for unstable hydrophobic protein environments while maintaining sequencing capability through fluorescent detection
Solution Approach 2:
The patent changes the fundamental parameter of the sequencing medium from protein-based hydrophobic environment to solid-state aqueous environment, improving manufacturing stability while preserving sequencing functionality
4Measurement precision
If polymerase is held closely to bottom of reaction chamber for sequencing-by-synthesis, then sequencing detection is enabled, but photodamage inactivates polymerase by photo-induced crosslinking
Solution Approach 1:
The patent extracts the polymerase from direct exposure to excitation light by using an opaque substrate that blocks light, thereby removing the cause of photo-induced crosslinking while preserving sequencing detection capability
Solution Approach 2:
The patent introduces an opaque substrate as an intermediary between the excitation light source and the reaction chamber, allowing light to reach fluorescent labels for detection while blocking light from reaching and damaging the polymerase
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 method provides a cost-effective and accurate sequencing process by blocking photodamage and enabling precise nucleotide identification, reducing the need for complex alignment and minimizing sequence gaps.
Implementation Method 1
an opaque metal layer that substantially blocks excitation light from penetrating into the reaction volume
Implementation Method 2
fluorescently labeled mononucleotides in the excitation zone emit fluorescent signals
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2
AI summary
The invention comprises a method for determining a nucleotide sequence of a polynucleotide. In some embodiments, the method comprises providing a solid state substrate comprising a cis side and a trans side. The fluorescently labeled polynucleotide strand comprises (i) a proximal end that is attached to the carrier particle, (ii) a distal end that is cleavable by an exonuclease, and (iii) at least one fluorescently labeled nucleotide comprising a fluorescent label. The trans side of the substrate is illuminated with excitation light to create a fluorescence excitation zone. While the substrate is illuminated, the fluorescently labeled polynucleotide strand is reacted with an exonuclease so that mononucleotides are released serially from the distal end of the strand and diffuse through the fluorescence excitation zone, so that fluorescently labeled mononucleotides in the excitation zone emit fluorescent signals. The fluorescent signals are detected as a function of time, enabling the deduction of a polynucleotide sequence.