Cleavable Nucleotide Analogs for DNA Sequencing
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Solution Overview
Problem
Current DNA sequencing methods using reversible terminators face limitations in improving DNA synthesis efficiency and accuracy with existing nucleotide analogs.
Innovation Solution
Development of cleavable nucleotide analogs with a cleavable moiety linked to the 3′-OH of the pentose, which includes protective groups, linkers, or linker lengthening moieties that can be attached to detectable moieties like fluorescent markers, allowing for reversible termination and restoration of the 3′-OH group for continued DNA synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If reversible terminators are used in DNA sequencing methods, then DNA synthesis can be controlled and terminated at specific positions, but the complexity of the nucleotide analog structure increases
Solution Approach 1:
The nucleotide analog is divided into distinct functional segments: a base moiety, a pentose ring, a cleavable moiety attached to the 3'-OH position, and a detectable moiety. This segmentation allows each component to perform its specific function independently while simplifying the overall design and synthesis of the reversible terminator.
Solution Approach 2:
The cleavable moiety acts as an intermediary between the 3'-OH group and the detectable moiety. It provides a reversible blocking function that can be selectively removed, enabling controlled termination and continuation of DNA synthesis while maintaining structural organization.
2Reliability
If cleavable moieties are attached to the 3'-OH of nucleotide analogs, then reversible termination is achieved, but the complexity of the sequencing process increases
Solution Approach 1:
The sequencing process employs periodic cycles of nucleotide incorporation, detection, and cleavage of the blocking moiety. Each cycle adds one nucleotide to the growing chain, allows detection of the incorporated base, then removes the blocking group to enable the next incorporation step, creating a reliable and repeatable process.
Solution Approach 2:
The cleavable moiety is pre-attached to the 3'-OH position before DNA synthesis begins. This preliminary attachment ensures that the reversible termination mechanism is already in place, allowing immediate control over chain elongation without requiring additional steps during the synthesis process.
3Measurement precision
If detectable moieties are attached to the nucleotide analog, then sequencing accuracy is improved, but the manufacturing complexity of the nucleotide analog increases
Solution Approach 1:
The detectable moiety is designed to serve multiple functions: it provides the signal for base identification, maintains the reversible termination capability through the attached cleavable group, and does not interfere with polymerase incorporation. This multi-functionality reduces the need for separate components and simplifies manufacturing.
Solution Approach 2:
The detectable moiety is specifically positioned at the base position of the nucleotide analog, while the cleavable moiety is attached to the 3'-OH of the pentose ring. This localized placement ensures that each part of the molecule performs its specific function optimally without interfering with other components, simplifying the manufacturing process.
Data Source
AI summary
Cleavable nucleotide analogs are provided. The nucleotide analog includes a nucleotide molecule attached to a cleavable moiety wherein the cleavable moiety comprises a protective group and/or a linker attached to a fluorophore. The cleavable moiety is linked to the oxygen atom of the 3′-OH of the pentose of the nucleotide molecule. The nucleotide analogs can be used in making polynucleotide molecules using template independent polymerases. The nucleotide analogs can act as reversible terminators during DNA sequencing by synthesis. The cleavage of the cleavable moiety restores a free 3′-OH functional group allowing growth of the polynucleotide molecule. The general structures as well as proposed synthetic schemes for the nucleotide analogs are also provided.