Nanopore Sequencing With Guide-Sequence Strand Displacement
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Solution Overview
Problem
Existing nanopore sequencing technologies face challenges such as complicated construction processes, low yield, strict preservation requirements, and the formation of secondary structures by nascent strands, which hinder efficient sequencing.
Innovation Solution
A method involving a sequencing library with a target double-stranded polynucleotide and primers that form unpaired regions, utilizing a polymerase with strand displacement activity to displace one strand through a nanopore while capturing electrical signal changes for sequencing, and a kit containing adapters, primers, and buffers for stable sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If a Y-shaped sequencing adapter complex is used to control nucleic acid conversion, then the single-stranded nucleic acid passes through the nanopore at a stable speed, but the construction process becomes complicated and the yield decreases
Solution Approach 1:
The patent divides the sequencing adapter into two separate simple adapters instead of using a complex Y-shaped adapter. The first adapter contains a guide sequence and the second adapter contains a complementary sequence. These separate adapters are easier to construct and combine, reducing construction complexity while maintaining the ability to control nucleic acid passage speed through the nanopore.
Solution Approach 2:
The patent extracts the helicase enzyme from the adapter structure itself, using a free-floating helicase enzyme instead of incorporating it into the Y-shaped adapter complex. This separation simplifies the adapter construction while the helicase enzyme independently performs the function of unwinding double-stranded nucleic acid at a controlled rate.
2Speed
If a Y-shaped sequencing adapter complex is used, then stable sequencing speed is achieved, but the preservation conditions become strict and long-term storage becomes difficult
Solution Approach 1:
By separating the adapter into two simple components rather than using a complex Y-shaped structure with embedded helicase, the patent creates adapters that are more stable and easier to store. The simple adapter sequences can be preserved under standard conditions, while the helicase enzyme is added separately during the sequencing process, eliminating the need for strict preservation conditions.
3Productivity
If polymerase-nucleic acid complex is used for nascent strand synthesis, then continuous synthesis proceeds, but the nascent strand forms complicated secondary structures that hinder nanopore capture
Solution Approach 1:
The patent introduces a guide sequence in the first adapter that acts as an intermediary to guide the nascent strand through the nanopore. This guide sequence prevents the nascent strand from forming complicated secondary structures by providing a defined path and interaction interface with the nanopore, ensuring efficient capture and sequencing while maintaining continuous synthesis productivity.
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
The method enables simple construction, long-term storage, and stable sequencing by controlling the unwinding and synthesis rate of double-stranded nucleic acids, preventing secondary structure formation and ensuring efficient sequencing.
Implementation Method 1
under an electric field force, the guide sequence is captured by the nanopore embedded in the membrane and passes through the nanopore
Implementation Method 2
detecting the electrical signal changes generated as the first strand passes through the nanopore during extension, to determine sequence information
Data Source
Figure 1(1)~1(5)
Figure 2(1)~2(6)
Figure 3(1)~3(6)
AI summary
Provided are a nanopore sequencing method and a kit. The method comprises: 1) providing a sequencing library, comprising target polynucleotide double strands, wherein the target polynucleotide double strands comprise a first strand and a second strand, and a 5' end of the first strand and a 3' end ofthe second strand comprise non-paired first single-stranded regions, which comprise a guide sequence and a second primer pairing region, respectively; 2) incubating a first primer and the sequencing library to for a sequencing complex, wherein the first primer has a 3' free end and is attached, by means of a 5' end, to a membrane embedded with a nanopore; 3) making a polymerase with strand displacement activity extend, by using the second strand as a template, the first primer, wherein under the action of an electric field force, the guide sequence is captured by the nanopore on the membrane and passes through the nanopore, and as the extension proceeds, the first strand is obtained by displacement and paases through the nanopore; and 4) detecting an electric signal change generated when the first strand passes through the nanopore in the extension process, and determining sequence information of the first strand of the target polynucleotide double strands.