Single-Stranded Nucleic Acid Construction for Nanopore Sequencing
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Solution Overview
Problem
Current methods for nucleic acid sequencing, such as those involving double-stranded molecules with hairpin loops, struggle with precision in detecting mutations and sequence errors, especially when analyzing single-stranded nucleic acids using nanopore sequencing, as they require repeated sequencing and have low efficiency in constructing molecules with correctly bound hairpin loops.
Innovation Solution
A method for constructing a single-stranded nucleic acid molecule using hairpin primers with a higher Tm value for the stem part than the single-stranded region, allowing repeated interrogation of the target sequence without complementary strand information, enhancing precision by synthesizing complementary strands that form hairpin structures and using λ exonuclease for decomposition, and incorporating adapter molecules with hairpin loop structures for strand displacement reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If double-stranded molecules with hairpin loops are used for sequencing, then sequencing can be performed, but precision in detecting mutations and sequence errors deteriorates
Solution Approach 1:
The invention separates the target sequence from its complementary strand, creating a single-stranded nucleic acid molecule that contains only the target sequence repeated multiple times. This segmentation eliminates the confusion between complementary strand information and actual mutations, allowing precise detection of mutations present in small quantities such as those from cancer cells.
Solution Approach 2:
Instead of sequencing double-stranded molecules and trying to distinguish mutations from complementary strand information, the invention inverts the approach by constructing single-stranded molecules that contain only the target sequence. This inversion fundamentally changes the sequencing paradigm to eliminate the source of error rather than trying to resolve it after sequencing.
2Measurement precision
If circular molecules with hairpin loops are used, then sequencing errors can be reduced by repeated sequencing, but the molecules cannot be analyzed by nanopore sequencing methods
Solution Approach 1:
The invention applies local quality by creating a single-stranded molecule with a specific structure: a target sequence region repeated multiple times (5-50 times) flanked by adapter sequences. This localized structural design allows the molecule to be compatible with nanopore sequencing while enabling repeated interrogation of the target sequence for high-precision mutation detection.
Solution Approach 2:
The invention transitions from two-dimensional double-stranded structures to a one-dimensional single-stranded structure with repeated sequences. This dimensional change allows the target sequence to be interrogated multiple times in a single linear molecule, achieving high precision without requiring circular structures that are incompatible with nanopore sequencing.
3Ease of manufacture
If ligation reaction is performed to form hairpin loop, then molecule construction can be achieved, but efficiency of constructing molecules with correctly bound hairpin loops deteriorates
Solution Approach 1:
The invention extracts the hairpin loop structure from the target sequence region and places it only in the adapter sequences at the ends of the molecule. This extraction eliminates the need for ligation reactions to form hairpin loops at the target sequence, significantly improving construction efficiency while maintaining the ability to perform repeated sequencing.
Solution Approach 2:
The invention uses copying by synthesizing the target sequence multiple times (5-50 times) in a linear array within the single-stranded molecule. This copying approach, achieved through PCR amplification with specific primers, is more efficient than ligation and allows high-fidelity reproduction of the target sequence for repeated interrogation during sequencing.
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 highly precise analysis of nucleic acid sequences by repeatedly interrogating the target sequence, improving detection of mutations present in small quantities, such as those from cancer cells, with a final determination precision of 99.9%, reducing errors and increasing efficiency compared to traditional methods.
Implementation Method 1
a hairpin primer including a single-stranded region at the 3′ terminal and a primer in a pair with the hairpin primer are used to synthesize a complementary strand of a template DNA
Implementation Method 2
synthesize a complementary strand of a template DNA including a target sequence
Implementation Method 3
the synthesized complementary strand forms a hairpin structure inside a molecule
Implementation Method 4
using λ exonuclease for decomposition
Data Source
AI summary
The present invention provides a method for constructing a single-stranded nucleic acid molecule for nucleic acid sequencing by means of a nanopore sequencer, said method including: a step in which at least one hairpin primer including a single-stranded region on the 3′ side and a pair of primers are used to synthesize a complementary strand of template DNA that includes the target sequence; and a step in which the synthesized complementary strand forms a hairpin structure inside a molecule and a template extension reaction is carried out. The obtained nucleic acid molecule includes both the target sequence and the complementary strand thereof in the sequence. Single strand construction enables analysis by nanopore sequencing, and the sequence of only the target nucleic acid, which does not include information of the complementary strand, is repeatedly analyzed, thus enabling analysis to be conducted with greater precision by addressing the problem of sequence errors.