Microwell Array Chip for Single Molecule Sequencing
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Solution Overview
Problem
Current gene sequencing methods are costly, time-consuming, and require complex processes, with limitations in detecting single molecules or cells, and are not suitable for genetic diagnosis of monogenic diseases due to high background noise and sequencing costs.
Innovation Solution
A method for gene sequencing using single molecule library preparation on a microwell array chip, where DNA fragments are amplified by PCR on the same chip, simplifying the process, reducing reagent use, and sequencing time and cost, by utilizing a semiconductor chip with microwells and ion-sensitive sensors to detect base incorporation during sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current sequencing methods are used, then sequencing can be performed, but the cost is high and the process is complex
Solution Approach 1:
The patent merges library preparation and sequencing operations onto a single microwell array chip. The chip integrates microfluidic channels for reagent delivery, PCR amplification zones, and sequencing detection areas, eliminating the need for separate devices and manual transfer steps between different instruments.
Solution Approach 2:
The microwell array chip serves multiple functions: it performs DNA library preparation, PCR amplification, and sequencing detection all in one device. The chip can handle multiple samples simultaneously in parallel microwells, making it a universal platform for various sequencing applications.
2Measurement precision
If current sequencing methods are used, then sequencing can be performed, but the time required is long
Solution Approach 1:
The patent implements continuous automated operations from library preparation through PCR amplification to sequencing detection without interrupting the sample. Reagents are continuously delivered through microfluidic channels, and the sequencing process begins immediately after amplification, eliminating idle time between steps.
Solution Approach 2:
The chip performs preliminary library preparation and PCR amplification steps automatically before sequencing begins. Adapters are pre-attached to DNA fragments, and amplification is completed in-situ on the chip, so that sequencing can start immediately without additional preparation time.
3Measurement precision
If current sequencing methods are used, then sequencing can be performed, but reagent consumption is high
Solution Approach 1:
The patent segments the sequencing process into individual microwell reactions, each handling a single DNA molecule or small number of molecules. This segmentation allows precise control of reagent volumes in each microwell, reducing overall reagent consumption compared to bulk processing methods.
Solution Approach 2:
The chip delivers reagents locally to specific microwells containing target DNA through microfluidic channels. This localized delivery ensures reagents are applied only where needed, minimizing waste and reducing total reagent consumption while maintaining sufficient concentration for accurate sequencing.
4Quantity of substance
If single molecule sequencing is used, then low amount of initial material is required, but background noise is high and accuracy is reduced
Solution Approach 1:
The patent performs preliminary PCR amplification of the single DNA molecule within the microwell before sequencing detection. This amplification step generates sufficient signal strength from the initial single molecule while maintaining the benefits of low starting material requirements.
Solution Approach 2:
The patent uses PCR amplification as an intermediary step between the single DNA molecule and the sequencing detection. The amplification process creates multiple copies that enhance the signal for detection, reducing background noise while still allowing the process to start from a single molecule.
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 simplifies the sequencing process, reduces costs, and improves sequencing efficiency by enabling single molecule library amplification and sequencing on a single chip, avoiding complex devices and reagents, while enhancing accuracy and reducing background noise.
Implementation Method 1
the released hydrogen ions or pyrophosphate PPi ions or increased charges of DNA backbones result in signal response of a sensor at bottom of the microwell
Implementation Method 2
the dNTP capable of pairing with a base under sequencing of the single-stranded DNA molecule as a template is ligated to the 3′ terminal of the sequencing primer S2 molecule in the presence of the sequencing enzyme, with hydrogen ions or pyrophosphate PPi ions released
Implementation Method 3
adding a mixture solution of DNA fragments to be tested in combination with a PCR amplification solution into the microwell array chip, allowing each micrawell on the micrawell array chip to contain the mixture solution, and sealing the micrawell array chip by a sealing cover on its surface, such that micrawells on the micrawell array chip each individually form reaction spaces
Data Source
AI summary
Provided is a method of gene sequencing on a microwell array chip, including: Step 1: adding a PCR amplification system containing DNA fragments to be sequenced into the microwell array chip, allowing microwells to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell; Steps 2 to 3: subjecting the microwell array chip to PCR amplification, and denaturing amplified double-stranded DNAs in individual microwells; and Steps 4 and 5: sequencing the DNA fragments with sequencing primer S2 molecules and dNTPs in each microwell with a sensor at the bottom of the microwell.