Universal Nucleic Acid Probe Reduces Sequencing Cost
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Solution Overview
Problem
Current nucleic acid sequencing by ligation methods are costly due to the large number of probes required, necessitate frequent primer replacement, and are sensitive to silver ions, which can cause precipitation and mismatch reactions.
Innovation Solution
A nucleic acid sequencing method using only four groups of probes, where each cycle adds one base to the sequencing chain without needing primer replacement, and employs a rapid excision method that minimizes damage to the sequencing system, avoiding silver ions by using alternative cleavage methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing-by-ligation methods use multiple groups of probes (e.g., 8 groups for 8 bases or 16 groups for SOLiD), then sequencing coverage is improved, but the cost increases significantly
Solution Approach 1:
The patent uses only four groups of probes (A, T, C, G) that can universally identify any base at the first position through fluorescent labeling. The same four groups are reused in subsequent cycles after primer extension, making the probe set multi-functional and eliminating the need for multiple different probe groups as in conventional methods.
Solution Approach 2:
The patent employs a reversible terminator mechanism where the 3′-OH group is blocked during ligation and then recovered in the next cycle after the blocked base is excised. This allows the same four groups of probes to be reused across multiple sequencing cycles, significantly reducing probe consumption and cost while maintaining complete sequencing coverage.
2Measurement precision
If sequencing methods require frequent primer replacement (e.g., every 8 bases or 5 bases in SOLiD), then sequencing accuracy is maintained, but time cost and operational complexity increase
Solution Approach 1:
The patent enables continuous sequencing by extending the primer by one base each cycle and immediately using it as the template for the next ligation reaction. The reversible terminator is removed and the 3′-OH is recovered, allowing the same primer to be used throughout the entire sequencing process without interruption or replacement, maintaining accuracy while eliminating time loss from primer changes.
3Productivity
If silver ions are used for excision in SOLiD method, then universal bases can be cleaved effectively, but harmful side reactions occur (precipitation with chloride ions and mismatch formation with T bases)
Solution Approach 1:
The patent uses a disposable chemical group (such as allyl, azide, or cyanovinyl) attached to the 3′-OH of the incorporated base that can be selectively removed by specific reagents. These chemical groups serve as temporary, single-use markers that are excised after detection, replacing the need for silver ions and avoiding all associated harmful side reactions while maintaining excision efficiency.
Solution Approach 2:
The patent introduces a chemical intermediary group between the base and the 3′-OH that mediates the excision process. This intermediary group (allyl, azide, or cyanovinyl) can be selectively cleaved by specific reagents without affecting other components in the system, avoiding the harmful interactions that occur with silver ions while still enabling effective removal of the terminator.
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 significantly reduces costs, maintains high sequencing accuracy, and improves read quality by reducing probe numbers and eliminating silver ion-related issues.
Implementation Method 1
a fluorescently modified DNA probe is ligated by a ligase
Implementation Method 2
the signal of the ligated probe is detected by an optical means
Implementation Method 3
Silver ions are used for excision (silver ions can specifically be bound with sulfur elements to cleave S—P bond)
Implementation Method 4
the template and primers are complementarily paired
Data Source
AI summary
A nucleic acid probe and a nucleic acid sequencing method for performing sequencing while ligating nucleic acids. The nucleic acid probe is a DNA sequencing probe, comprising a first moiety, a second moiety, a linker, and a detectable label. A base of the first moiety is A, T, U, C, or G, a base of the second moiety is a random base and/or a universal base, and 3 bases or more are present in the second moiety. The first moiety and the second moiety are ligated via the linker, the connection between the first moiety and the ligation can be cleaved, and the detectable label is ligated to the second moiety or the linker. The above probe, a combination formed therewith, or a sequencing method using the same can reduce the number or types of probes in nucleic acid sequencing, thereby reducing cost.