Nucleic Acid Sequencing Under Tension via Length Detection
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Solution Overview
Problem
Current methods for sequencing nucleic acids under tension lack efficiency in detecting nucleotide incorporation without the use of extrinsic labels and are limited by the need for physical separation of polymerase-mediated extension reactions.
Innovation Solution
The method involves immobilizing nucleic acids under tension and detecting nucleotide incorporation by measuring changes in length using a tethered observable moiety, such as a bead, without the need for fluorescence detection, and performing sequencing-by-synthesis reactions using a polymerase in the presence of natural or modified nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing methods use extrinsic labels and physical separation of polymerase-mediated extension reactions, then detection accuracy is improved, but device complexity and manufacturing complexity increase
Solution Approach 1:
The patent extracts and eliminates the need for extrinsic labels (such as fluorescent dyes) and physical separation mechanisms from the sequencing system. By detecting nucleotide incorporation through intrinsic length changes of the nucleic acid itself, the method removes complex labeling and separation components while maintaining detection accuracy.
Solution Approach 2:
The nucleic acid molecule serves itself as both the template and the detectable element. The polymerase-mediated extension reaction naturally produces length changes that can be detected without requiring external labels or physical separation mechanisms. The system uses the inherent mechanical properties of the nucleic acid to provide the detection signal.
2Measurement precision
If physical separation of polymerase-mediated extension reactions is used, then measurement precision is improved, but productivity decreases
Solution Approach 1:
The patent merges multiple polymerase-mediated extension reactions into a single simultaneous process. Instead of physically separating reactions to improve measurement precision, the method allows multiple reactions to occur concurrently and detects them through their collective length changes, thereby maintaining precision while dramatically increasing productivity.
Solution Approach 2:
The detection system is designed to universally detect length changes from multiple simultaneous polymerase reactions without requiring separate detection channels for each reaction. This multi-functional detection approach enables parallel processing of multiple templates, enhancing throughput while maintaining measurement precision.
3Measurement precision
If extrinsic labels are used for detection, then measurement precision is improved, but loss of substance and device complexity increase
Solution Approach 1:
The patent removes extrinsic labels from the sequencing system entirely. By detecting the intrinsic length changes of the nucleic acid during polymerase-mediated extension, the method eliminates the need for fluorescent dyes, radioactive isotopes, or other extrinsic labeling substances, thereby avoiding associated loss and complexity issues.
Solution Approach 2:
The nucleic acid provides its own detection signal through intrinsic length changes during synthesis. This self-service mechanism eliminates the need for external labels that would otherwise be required for detection, reducing substance loss and simplifying the overall system.
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-speed, high-throughput sequencing of nucleic acids by detecting nucleotide incorporation through length changes, allowing for simultaneous sequencing of multiple templates without the requirement for extrinsic labels or physical separation of reactions, thereby improving sequencing efficiency and accuracy.
Implementation Method 1
incorporation of a nucleotide into a newly synthesized nucleic acid hybridized to the template nucleic acid, and consequently upon conversion of a single stranded region to a double stranded region of the template nucleic acid
Implementation Method 2
DNA has been stretched using electrical forces (Schurr et al. Biopolymers 29, 1161-1165 (1990)), electrophoresis (Smith et al. Biopolymers 1990, 1167-73 (1990))
Implementation Method 3
when stretched by relatively low forces (e.g., less than or equal to 6 picoNewtons (pN)), single stranded DNA (ssDNA) is more compact than double stranded DNA (dsDNA)
Data Source
AI summary
The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.