Cleavable Linkers for DNA Sequencing Read Length
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Solution Overview
Problem
Current DNA sequencing methods using cleavable fluorescent nucleotide reversible terminators are limited by the need for additional capping steps, which increase process complexity and restrict the sequencing read length to only 10 bases due to the presence of a long remnant tail after disulfide linkage cleavage.
Innovation Solution
Development of nucleotide analogues with a deoxyribose or ribose, a base attached to the 1' position, a blocking group on the 3' oxygen, and a detectable label bound via a cleavable linker, allowing for orthogonal chemically cleavable linkers that enable longer read lengths by stabilizing the linker during sequencing reactions and avoiding extra capping steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If disulfide linkage is used to attach fluorescent dye to the base, then the nucleotide can be incorporated and detected, but the sequencing read length is limited to only 10 bases due to the long remnant tail after cleavage
Solution Approach 1:
The patent changes the chemical structure of the cleavable linker from disulfide to alternative linkers (azo, nitrobenzyl, allyl, dimethyl ketal, azidomethyl) that produce shorter or no remnant tails after cleavage. This structural parameter change directly resolves the limitation of long remnant tails that restricted read length to 10 bases, enabling longer sequencing reads by eliminating the harmful residual structure.
2Measurement precision
If disulfide linkage cleavage is used to remove the fluorescent label, then the base identity can be determined, but an additional capping step with alkylating agent is required
Solution Approach 1:
The patent extracts and removes the problematic disulfide linkage system that required additional capping steps. By replacing it with alternative cleavable linkers (azo, nitrobenzyl, allyl, dimethyl ketal, azidomethyl) that cleave cleanly without leaving reactive groups, the patent eliminates the need for the extra capping step, thereby reducing process complexity while maintaining base identification accuracy.
Solution Approach 2:
The patent employs disposable alternative linkers that perform their function (attaching and releasing the fluorescent label) and then are cleanly removed without requiring further processing. These linkers are designed to be used once and discarded after cleavage, unlike the disulfide system that required additional reagents (iodoacetamide) to cap the resulting thiol groups, thus simplifying the overall process.
3Productivity
If disulfide linkage is used to attach the fluorescent dye, then the nucleotide can serve as a reversible terminator, but the long remnant tail limits the addition of multiple nucleotides in a row
Solution Approach 1:
The patent changes the chemical parameters of the cleavable linker to produce shorter or negligible remnant tails. By using alternative linkers (azo, nitrobenzyl, allyl, dimethyl ketal, azidomethyl) instead of disulfide linkers, the patent enables multiple nucleotides to be added in succession without the accumulation of long remnant tails, thereby increasing productivity and extending readable sequence length.
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 allows for more stable and efficient DNA sequencing with longer read lengths, reducing process complexity and improving the accuracy of nucleotide identification by using orthogonal cleavable linkers that maintain the 3' OH group's integrity for subsequent cycles.
Implementation Method 1
a detectable label bound to the base via a cleavable linker... different types of linkers (e.g., disulfide, Azo, nitrobenzyl, allyl, dimethyl ketal, or azidomethyl)
Implementation Method 2
removal of the blocking group to regenerate the 3' OH group... different types of linkers (e.g., disulfide, Azo, nitrobenzyl, allyl, dimethyl ketal, or azidomethyl)
Data Source
AI summary
Disclosed herein, inter alia, are nucleotide analogues, and methods of use thereof, having cleavable orthogonal linkers.