Reversible Extension Terminator Sequencing via Optical Confinement
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Solution Overview
Problem
Current DNA sequencing methods, such as Sanger's dideoxy termination and Maxam-Gilbert's chemical degradation, are labor-intensive and time-consuming due to the need for washing steps between base incorporation events, which delays the sequencing process and depletes costly reagents.
Innovation Solution
The use of reversible extension terminators in conjunction with optical confinements allows for the detection of nucleotide incorporation without the need for washing, enabling continuous sequencing by incorporating nucleotide analogs with removable blocking groups that can be detected and reused, thereby eliminating the rate-limiting washing step.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If washing steps are performed between base incorporation events, then unincorporated nucleotides are removed, but the sequencing process is delayed and costly reagents are depleted
Solution Approach 1:
The patent extracts the detection function from the washing step by using optical confinements to detect incorporated nucleotides in situ, eliminating the need to physically remove unincorporated nucleotides through washing. This allows continuous sequencing without the productivity loss from washing steps while maintaining detection precision through optical methods.
Solution Approach 2:
The patent introduces an optical intermediary (light/detector system) that mediates between the incorporated nucleotide and the detection system, allowing identification of base incorporation events without physical separation or washing steps, thus resolving the contradiction between accurate detection and sequencing speed.
2Measurement precision
If washing steps are performed between base incorporation events, then unincorporated nucleotides are removed, but costly reagents are depleted
Solution Approach 1:
The patent extracts the detection function from the washing step by using optical confinements to detect incorporated nucleotides in situ, eliminating the need to physically remove unincorporated nucleotides through washing. This allows continuous sequencing without the productivity loss from washing steps while maintaining detection precision through optical methods.
Solution Approach 2:
The optical confinement system provides self-service detection by automatically identifying incorporated nucleotides through their optical properties without requiring external washing or separation steps, thereby preventing reagent depletion while maintaining detection accuracy.
3Productivity
If reversible extension terminators are used without washing, then sequencing speed is improved, but detection of incorporated nucleotides becomes more challenging
Solution Approach 1:
The patent introduces an optical intermediary (light/detector system) that mediates between the incorporated nucleotide and the detection system, allowing identification of base incorporation events without physical separation or washing steps, thus resolving the contradiction between accurate detection and sequencing speed.
Solution Approach 2:
The patent exploits color changes (fluorescence emission at specific wavelengths) of incorporated nucleotides as optical signatures that can be detected without washing. Each nucleotide type emits characteristic fluorescence, enabling unambiguous identification directly in the reaction mixture, thus facilitating fast washing-free sequencing while maintaining detection accuracy.
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 high-throughput and cost-effective nucleic acid sequencing by reducing the need for reagent depletion and streamlining the sequencing process, allowing for faster and more efficient DNA analysis.
Implementation Method 1
The nucleotide analogs used in the present invention comprise detectable labels, such as fluorescent labels
Implementation Method 2
the first nucleotide analog comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the first nucleotide analog is incorporated into the nascent nucleic acid strand
Data Source
AI summary
The present invention relates to the uses of reversible extension terminator in conjunction with optical confinements in nucleic acid sequencing. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost nucleic acid sequencing.