Total Reflection Microscope Nucleic Acid Sequencing System
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Solution Overview
Problem
Current gene sequencing technologies face challenges such as complex and time-consuming library construction, PCR amplification biases, short sequencing reads, and high error rates in nanopore sequencing, limiting their efficiency and scalability.
Innovation Solution
A nucleic acid sequencing system incorporating a base with a clamping platform, reagent storage, fluid control, and a total reflection microscope for single-molecule fluorescence sequencing, which eliminates the need for library construction and PCR amplification, using dual-bandpass optical filters and dichroscopes to focus lasers and detect fluorescence for direct DNA/RNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If second-generation high-throughput sequencing technology is used, then sequencing throughput is improved, but library construction complexity and time increase
Solution Approach 1:
The invention extracts and eliminates the library construction step from the sequencing workflow. By using a microfluidic chip that directly sequences DNA fragments without requiring prior library preparation, the system removes this complex and time-consuming preprocessing step while maintaining high sequencing throughput
Solution Approach 2:
The invention segments the sequencing process into independent micro-reactions occurring in parallel within the microfluidic chip. Multiple DNA fragments are sequenced simultaneously in separate micro-chambers, enabling high throughput without requiring complex library construction procedures
2Illumination intensity
If PCR amplification is performed in second-generation sequencing, then signal intensity is improved, but sequencing bias and distortion of original gene proportion occur
Solution Approach 1:
The invention uses self-labeling nucleotides that automatically incorporate fluorescent labels during the sequencing reaction itself, eliminating the need for separate PCR amplification steps. This self-service approach maintains signal intensity while avoiding PCR-induced bias and distortion of gene proportions
Solution Approach 2:
The invention replaces the mechanical PCR amplification process with a biochemical sequencing-by-synthesis approach using fluorescently labeled nucleotides. This substitution eliminates the need for thermal cycling and DNA amplification, directly sequencing native DNA molecules to preserve original gene proportions
3Device complexity
If nanopore sequencing technology is used, then device simplicity is improved, but sequencing error rate increases due to weak electrical signals
Solution Approach 1:
The invention replaces the electrical detection method of nanopore sequencing with optical detection using fluorescence microscopy. This substitution maintains device simplicity while dramatically improving signal strength and reducing error rates by using photon detection instead of weak electrical signal measurement
Solution Approach 2:
The invention changes the detection parameter from electrical current (picoamp/nanoamp level) to optical fluorescence intensity. This parameter change maintains the simplicity of direct single-molecule sequencing while providing a much stronger and more reliable signal for accurate base calling
4Productivity
If second-generation sequencing is used, then sequencing throughput is improved, but read length decreases making bioinformatics analysis difficult
Solution Approach 1:
The invention uses a dynamic imaging approach where the microfluidic chip can be moved relative to the microscope field of view, allowing sequential imaging of different regions. This dynamic capability enables capture of long-range information across the entire chip while maintaining high throughput through parallel processing
Solution Approach 2:
The invention adds the spatial dimension to the sequencing process by using a two-dimensional array of micro-chambers on the chip that can be imaged in parallel. This dimensional approach allows long reads to be captured across multiple fields of view while maintaining high throughput through simultaneous imaging of multiple locations
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 system enables direct, efficient, and cost-effective sequencing of DNA/RNA molecules with simple operation, suitable for clinical applications, by leveraging single-molecule fluorescence technology and total internal reflection fluorescence microscopy, reducing sequencing errors and increasing read lengths.
Implementation Method 1
the micro objective is configured to focus the lasers on the gene sequencing chip with an incident angle greater than a critical angle, so as to excite a sample in the gene sequencing chip to produce fluorescence
Implementation Method 2
single-molecule fluorescence sequencing technology, the total internal reflection fluorescence microscopy
Data Source
AI summary
A nucleic acid sequencing system is provided, including a base provided with a clamping platform, a reagent storage unit, a fluid control unit, a mobile platform and a total reflection microscope thereon; the clamping platform being provided with a gene sequencing chip thereon; the reagent storage unit being configured to store a gene sequencing reagent, the fluid control unit being configured to pump the gene sequencing reagent from the reagent storage unit to the gene sequencing chip, the mobile platform being configured to drive the clamping platform to move toward or away from the total reflection microscope; and the total reflection microscope being configured to detect a gene sequence of a sample in the gene sequencing chip.