Nucleic Acid Sequencing Kit Using Segmented Probes and Reversible Blocking
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Solution Overview
Problem
Current nucleic acid sequencing methods, such as sequencing-by-ligation and sequencing-by-synthesis, face challenges including high costs, inefficient sequencing reads, and the presence of scar atoms that affect accuracy and require costly modifications, leading to increased sequencing time and costs.
Innovation Solution
A method combining sequencing-by-ligation with reversibly blocked dNTP polymerization, which involves hybridizing a sequencing primer, ligating nucleic acid probes, detecting labels, and excising blocking groups to improve sequencing efficiency and accuracy, while reducing costs by minimizing the need for additional primers and avoiding scar atoms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing-by-ligation method is used with multiple probes and universal bases, then sequencing coverage is improved, but cost increases and sequencing time extends due to frequent primer reloading
Solution Approach 1:
The probe is divided into distinct functional segments: a fixed 5' segment containing the universal base and a 3' segment with the fluorescent label and random sequence. This segmentation allows the fixed portion to remain bound to the template while enabling efficient removal and replacement of the labeled segment, reducing primer reloading frequency
Solution Approach 2:
The probe is pre-designed with a universal base at the 5' end that complements a universal base in the primer, creating a stable initial binding. This preliminary action ensures that the probe remains anchored during sequencing cycles, eliminating the need for frequent primer reloading and reducing sequencing time
2Measurement precision
If sequencing-by-synthesis with reversibly blocked dNTP is used, then sequencing accuracy is improved, but cost increases due to modified nucleotides and scar atoms
Solution Approach 1:
The invention uses standard, unmodified dNTPs instead of expensive reversibly blocked dNTPs. The probe itself serves as the temporary carrier of the fluorescent label, and after sequencing, the probe is simply removed without leaving scar atoms. This approach replaces expensive modified nucleotides with cheaper, disposable probes
Solution Approach 2:
The invention changes the parameter of nucleotide modification from modified dNTPs to unmodified dNTPs. By keeping the nucleotides in their natural state and placing all modifications on the removable probe, the cost of sequencing is reduced while maintaining accuracy through the probe's fluorescent labeling
3Productivity
If universal bases and random sequences are used in probes, then ligation efficiency is improved, but scar atoms are generated that affect subsequent sequencing accuracy
Solution Approach 1:
The invention extracts the universal base and random sequence from the dNTP and places them on the probe instead. This extraction ensures that when nucleotides are incorporated, only standard bases are added to the sequence, preventing scar atom formation. The probe containing the universal base is removed after sequencing, leaving no interference
Solution Approach 2:
The probe acts as an intermediary that temporarily carries the universal base and fluorescent label during sequencing. The probe mediates the ligation reaction with its universal base complementary to the primer's universal base, enabling efficient ligation without requiring modified nucleotides that would leave scar atoms
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 enhances sequencing reads, accuracy, and reduces costs by eliminating the need for extensive primer reloading and minimizing scar atom interference, resulting in a more efficient and cost-effective sequencing process.
Implementation Method 1
a fluorescently modified DNA probe is ligated by a ligase
Implementation Method 2
a fluorescently modified DNA probe is ligated by a ligase
Implementation Method 3
a dNTP is ligated by a polymerase
Implementation Method 4
a dNTP is ligated by a polymerase
Implementation Method 5
the signal of the ligated probe is detected by an optical means to determine the sequence at that position
Data Source
AI summary
Provided are a nucleic acid sequencing method and a nucleic acid sequencing kit. The kit comprises a nucleic acid probe, a ligase, dNTP having a blocking group attached to a 3′ end, a polymerase, a reagent 1 for excising the blocking group attached to the 3′ end of the dNTP, and a reagent 2 for excising the remaining nucleotides on the nucleic acid probe that are not bound to a to-be-tested base group.