DNA Polymerase-Modified Single Molecule Device for Sequencing
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Solution Overview
Problem
Current DNA sequencing technologies face challenges such as long duration, high costs, complexity, and instability, particularly in controlling DNA movement speed and achieving accurate sequencing signals, which hinder rapid and cost-effective genetic analysis.
Innovation Solution
A single molecule device modified with DNA polymerase, utilizing graphene or silicon nanowires as electrodes, where an amino acid residue linked to the polymerase in a conformation-changing region allows for real-time electrical signal detection during DNA synthesis, enabling improved sensitivity and accuracy in sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nanopore-based electrical test platform is used for DNA sequencing, then the sequencing can be performed without PCR amplification, but the DNA movement speed control is difficult and sequencing signal resolution is low
Solution Approach 1:
The patent introduces DNA polymerase as an intermediary component that bridges the DNA template and the graphene electrode. The polymerase binds to the graphene surface through specific amino acid residues (such as cysteine), creating a stable complex that enables direct electrical signal detection of DNA synthesis without requiring PCR amplification or complex nanopore control mechanisms
Solution Approach 2:
The patent replaces the mechanical nanopore-based DNA translocation system with an electrochemical detection system using graphene electrodes. Instead of mechanically controlling DNA movement through nanopores, the system uses electrical signal detection to monitor DNA polymerase activity directly, achieving higher signal resolution and easier speed control
2Measurement precision
If PCR amplification is used in sequencing, then the signal detection is enhanced, but the time-consuming and cost increase significantly
Solution Approach 1:
The patent extracts and eliminates the PCR amplification step from the sequencing workflow. By using direct single-molecule detection with graphene-based electrodes and DNA polymerase, the system achieves sufficient signal detection sensitivity without requiring the time-consuming PCR amplification process, reducing sequencing duration while maintaining detection quality
Solution Approach 2:
The system enables self-service detection where the DNA polymerase itself generates the detectable electrical signal during DNA synthesis. The polymerase's natural catalytic activity is directly transduced into electrical signals by the graphene electrode, eliminating the need for external amplification services and reducing overall processing time
3Measurement precision
If fluorescence labeling method is used, then the sequencing signal can be detected, but the process complexity and instability increase
Solution Approach 1:
The patent substitutes the optical fluorescence detection system with an electrical detection system using graphene electrodes. Instead of using fluorescent labels and complex optical detection pathways, the system directly detects electrical signals from DNA polymerase activity, dramatically simplifying the detection process while improving stability and reducing procedural complexity
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 significantly enhances sequencing speed and accuracy by directly monitoring DNA polymerase activity, eliminating the need for PCR amplification and reducing errors, allowing for longer sequence detection in a single reaction.
Implementation Method 1
an amino acid residue providing the linkage site is located in a conformation changing region of the DNA polymerase... the change in the conformation of the DNA polymerase causes the change of the current in the single molecule device
Data Source
AI summary
Disclosed is a DNA sequencing method. The DNA sequencing method of this invention comprises: (1) adding a tag sequence at the 3′ terminus of the DNA to be sequenced so as to form a DNA to be sequenced including the tag sequence, the nucleotide sequence of said tag sequence being the reverse complement of the nucleotide sequence of the sequencing primer; (2) mixing the DNA to be sequenced including the tag sequence and the sequencing primer so as to form a product having a 5′ terminus double strand and a single main strand; (3) after step (2) is completed, the product is mixed separately with dATP, dCTP, dTTP, and dGTP to obtain four systems, each system is separately added to a single-molecule device modified by a DNA polymerase, and electric signals are read. Experiments verify that the method of this invention performs DNA sequencing and has important application value.