Nanopore Nucleic Acid Translocation via Single-Stranded Tails
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Solution Overview
Problem
Existing nanopore-based nucleic acid sequencing technologies face challenges in controlling DNA translocation rates, nucleotide discrimination, and sample preparation complexity, with trade-offs in device fabrication and detection sensitivity.
Innovation Solution
The method involves preparing nucleic acid analytes as double-stranded products with single-stranded tails that can be captured by nanopores, allowing controlled translocation and analysis by applying an electrical field, eliminating the need for nucleic acid-denaturing conditions and simplifying sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA translocation rate is increased to improve productivity, then sequencing throughput is improved, but nucleotide discrimination precision deteriorates
Solution Approach 1:
The DNA molecule is segmented into individual nucleotides that pass through the nanopore one at a time, allowing each nucleotide to be individually detected and identified by its unique electrical signature, thus maintaining measurement precision while enabling continuous high-speed sequencing
Solution Approach 2:
A motor protein is introduced as an intermediary that controls the translocation of DNA through the nanopore at a regulated pace, ensuring that each nucleotide is properly positioned and detected before the next one enters, thereby maintaining discrimination precision while enabling sustained productivity
2Ease of operation
If sample preparation is simplified to improve ease of operation, then device fabrication complexity is reduced, but detection sensitivity deteriorates
Solution Approach 1:
The system uses the DNA molecule's own structural properties and the nanopore's inherent ability to discriminate single-stranded from double-stranded DNA to achieve detection without requiring complex sample preparation steps, thus maintaining detection sensitivity while simplifying sample preparation
Solution Approach 2:
The system changes the physical state of DNA from double-stranded to single-stranded form, which fundamentally alters its interaction with the nanopore and enables direct detection without complex preparation, maintaining sensitivity while simplifying操作流程
3Productivity
If denaturing conditions are applied to improve nucleotide accessibility, then translocation control is improved, but sample preparation complexity increases
Solution Approach 1:
The DNA is pre-denatured into single-stranded form before loading into the nanopore system, which preliminary prepares the sample for optimal translocation and detection, improving nucleotide accessibility while avoiding the need for complex denaturing conditions during the actual sequencing process
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 reduces translocation speed to improve analysis efficiency without complicating sample preparation steps, enabling reliable nucleotide identification and simplified data analysis.
Implementation Method 1
capturing the 5′ non-complementary tail of the double stranded product by the nanopore by applying an electrical field across the nanopore
Implementation Method 2
translocating at a detectable rate the labeled extension strand of the captured double stranded product through the nanopore by the applied electrical field
Data Source
AI summary
The invention provides methods for analyzing polynucleotides using nanopores that allow passage of single stranded polynucleotides but not double stranded polynucleotides. In accordance with some embodiments, a double-stranded product is produced that comprises a labeled strand with a single stranded tail or overhang. The double stranded product is exposed to one or more nanopores in the presence of an electric field across the one or more nanopores such that the single stranded tail may be captured and the labeled strand translocated by unzipping from the double stranded product. The ionic composition of the reaction mixture and electric field strength are selected so that nucleotides translocate a nanopore at a rate of less than 1000 nucleotides per second.