Cleavable Linker Nucleotides for Sequencing Accuracy

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Solution Overview

Problem

Current DNA sequencing methods, particularly sequencing by synthesis (SBS), face challenges in accurately and efficiently incorporating modified nucleotides into growing DNA chains, which is crucial for achieving high-throughput and cost-effective genome sequencing.

Innovation Solution

The method involves using a reaction mixture containing four nucleotides, each labeled with a fluorophore moiety attached via a cleavable linker. The reaction is conducted with a polymerase and a primer hybridized to a target polynucleotide, where the incorporation of nucleotides is detected by exciting the fluorophores and monitoring the cleavage of the linkers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If fluorophore moieties are attached to all four nucleotides for detection, then sequencing accuracy is improved, but the complexity of the sequencing system increases

Engineering Contradiction:

Improvesequencing accuracyVSAvoidsystem complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The four nucleotides are divided into two groups based on their fluorophore attachment characteristics. Two nucleotides (dATP and dCTP) are attached via cleavable linkers that release fluorophores upon incorporation, while the other two nucleotides (dGTP and dTTP) use non-cleavable linkers that retain fluorophores. This segmentation allows differentiation of nucleotide identities through distinct fluorescence patterns, improving sequencing accuracy while managing system complexity through systematic categorization.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Different types of linkers (cleavable vs. non-cleavable) are applied to different nucleotide positions based on their specific identification needs. The cleavable linker group (dATP, dCTP) and non-cleavable linker group (dGTP, dTTP) create locally distinct chemical properties that enable differentiation. This local quality variation allows the system to encode nucleotide identity information through fluorescence behavior rather than requiring entirely different fluorophores for each nucleotide.

Inventive Principle:

Principle #3Local quality

2Measurement precision

If multiple cleavable linkers with different cleavage conditions are used, then nucleotide identification accuracy is improved, but the duration of the sequencing process increases

Engineering Contradiction:

Improvenucleotide identification accuracyVSAvoidsequencing process duration

Core Design Contradiction:

Measurement precisionVSDuration of action of moving object

Solution Approach 1:

The sequencing process employs periodic cycles of fluorophore excitation, detection, and selective cleavage. Each cycle incorporates nucleotides and then applies specific cleavage conditions (e.g., reducing agents for disulfide linkers, hydroxylamine for hydrazone linkers) to release fluorophores from specific nucleotide groups. This periodic action allows systematic differentiation of nucleotide identities through repeated measurement-cleavage cycles, improving identification accuracy while maintaining a structured process duration.

Inventive Principle:

Principle #19Periodic action

Solution Approach 2:

Different types of cleavable linkers serve as intermediaries between the fluorophore and the nucleotide base. These linkers (disulfide, hydrazone, etc.) act as chemical mediators that can be selectively cleaved under specific conditions to release fluorophores from particular nucleotide groups. The intermediary linkers enable controlled, condition-specific fluorophore release, allowing accurate nucleotide identification through differential cleavage patterns without requiring simultaneous processing of all nucleotides.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If cleavable linkers are used to enable fluorophore removal, then sequencing throughput is improved, but the reliability of the nucleotide incorporation detection decreases

Engineering Contradiction:

Improvesequencing throughputVSAvoiddetection reliability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The nucleotide pool is segmented into cleavable and non-cleavable groups, where cleavable nucleotides (dATP, dCTP) allow fluorophore removal after detection to enable subsequent cycles, while non-cleavable nucleotides (dGTP, dTTP) maintain permanent fluorophore attachment. This segmentation ensures that at least some nucleotides provide reliable, persistent fluorescence signals for detection, maintaining detection reliability while enabling throughput improvement through cleavable linker removal in subsequent cycles.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The system employs feedback mechanisms where the fluorescence detection results from one cycle inform the cleavage conditions applied in subsequent cycles. The detection of which fluorophores are released (or retained) provides feedback about which nucleotides were incorporated, allowing the system to adjust and optimize cleavage conditions to maintain reliable detection while maximizing throughput through efficient fluorophore management.

Inventive Principle:

Principle #23Feedback

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 accurate identification and incorporation of nucleotides, facilitating high-throughput DNA sequencing by synthesizing the DNA strand while providing real-time detection of incorporated bases.

Implementation Method 1

a first fluorophore moiety that generates a first emission light when excited by a first excitation light and a second fluorophore moiety that generates a second emission light when excited by a second excitation light

Methodology Applied

Scientific EffectFluorescence: Fluorescence