MspA Nanopore Tunnel for Rapid Low-Input DNA Sequencing
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Solution Overview
Problem
Established DNA sequencing technologies require substantial amounts of DNA and several lengthy steps to construct just several tens of bases out of the full sequence, which is expensive and time-consuming, especially when sequencing mammalian genomes.
Innovation Solution
A method involving Mycobacterium smegmatis porin (Msp) porin with a vestibule and constriction zone is positioned between conductive liquid media, and mutant Msp porins with specific mutations are used to define a tunnel for analyte detection and sequencing, utilizing electric fields and molecular motors for translocation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If established DNA sequencing technologies are used, then sequencing can be performed, but substantial amounts of DNA and several lengthy steps are required, making it expensive and time-consuming
Solution Approach 1:
The invention divides the DNA molecule into individual base pairs that can be sequentially translocated through the nanopore one at a time, enabling rapid detection of each base without requiring lengthy construction steps. The nanopore acts as a segmentation point where the continuous DNA strand is processed base-by-base, dramatically increasing sequencing speed and reducing processing time from days to minutes.
2Productivity
If established DNA sequencing technologies are used, then sequencing can be performed, but substantial amounts of DNA are required
Solution Approach 1:
The invention extracts and utilizes only the essential functional element needed for sequencing - the nanopore itself - rather than requiring bulk DNA processing. By focusing on the pore's ability to detect individual bases as they pass through, the system achieves high sequencing efficiency with minimal DNA input, eliminating the need for substantial DNA quantities required by conventional methods.
3Productivity
If Msp porin with vestibule and constriction zone is used to define a tunnel, then efficient and rapid sequencing is enabled, but the device complexity increases
Solution Approach 1:
The Msp porin structure employs local quality by creating distinct functional zones within the protein: a vestibule region for initial analyte interaction and a constriction zone for precise base detection. This spatial differentiation of functional properties within the single porin molecule enables high-throughput sequencing while maintaining a relatively simple overall structure, as each zone performs its specific function without requiring additional complex components.
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
Facilitates efficient and rapid sequencing of nucleic acids with reduced DNA requirements and shorter processing times, enabling high-throughput analysis.
Implementation Method 1
a first conductive liquid medium and a second conductive liquid medium
Implementation Method 2
modifying the conductance through the tunnel of an Msp porin
Implementation Method 3
applying an electric field to a Mycobacterium smegmatis porin (Msp) porin
Data Source
AI summary
Provided herein are Mycobacterium smegmatis porin nanopores, systems that comprise these nanopores, and methods of using and making these nanopores. Such nanopores may be wild-type MspA porins, mutant MspA porins, wild-type MspA paralog porins, wild-type MspA homolog porins, mutant MspA paralog porins, mutant MspA homolog porins, or single-chain Msp porins. Also provided are bacterial strains capable of inducible Msp porin expression.