Thiol Cleavage Reagents for Sequencing Accuracy
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Solution Overview
Problem
Current nucleotide analogs for sequencing technologies, such as Sequencing by Synthesis (SBS), face challenges in achieving longer read lengths and higher accuracy due to reactive residues left after cleavage, which lead to reduced read lengths and increased error rates.
Innovation Solution
The development of deoxynucleoside triphosphates with a 3′-O position capped by a methylenedisulfide group as a cleavable protecting group and a detectable label connected via a cleavable oxymethylenedisulfide linker, which allows for reversible protection and clean removal without leaving reactive sulfur residues, along with the use of thiol-containing compounds as cleave agents and oxidative wash steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional cleavable protecting groups and linkers are used in nucleotide analogs for sequencing, then the sequencing process can proceed with label detection, but reactive sulfur residues are left after cleavage which reduce read length and increase error rates
Solution Approach 1:
The patent extracts and removes the harmful sulfur residues from the cleavage process by using non-sulfur containing protecting groups (acetyl, benzoyl, carbamates) and linkers instead of traditional disulfide-based groups. This extraction of the harmful element (sulfur) eliminates the source of reactive residues while maintaining the essential functionality of reversible protection and cleavage for sequencing accuracy.
Solution Approach 2:
The patent changes the chemical parameters of the protecting groups and linkers from sulfur-containing (disulfide) to non-sulfur-containing (acetyl, benzoyl, carbamate) groups. This parameter change in chemical composition fundamentally alters the cleavage products from reactive sulfur residues to non-reactive byproducts, thereby improving sequencing accuracy and extending read length without compromising the reversibility of protection.
2Duration of action of moving object
If traditional disulfide-based protecting groups are used, then reversible protection can be achieved, but side reactions occur due to reactive residues that limit read length
Solution Approach 1:
The patent converts the potentially harmful reactive sulfur residues into beneficial non-reactive cleavage products by selecting alternative protecting groups and linkers. The acetyl, benzoyl, and carbamate groups provide clean cleavage without generating reactive species, thereby extending the duration of accurate sequencing (read length) while maintaining low error rates. The harmful aspect is transformed into a beneficial clean cleavage process.
3Measurement precision
If cleavable linkers with sulfur residues are used for label attachment, then label detection is enabled, but sulfur residue-related errors increase
Solution Approach 1:
The patent extracts sulfur from the linker chemistry by using non-sulfur containing linkers such as acetyl, benzoyl, and carbamate-based linkers. This extraction eliminates the source of sulfur residue errors while preserving the essential function of reversible label attachment and detection, thereby maintaining detection accuracy without the harmful side effects of sulfur-containing cleavage products.
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 accuracy and read length by preventing side reactions and sulfur residue-related errors, improving the overall performance of sequencing technologies like SBS.
Implementation Method 1
wherein said cleave reagent comprises a thiol-containing compound; subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, wherein said first deoxynucleoside triphosphate is incorporated (into the primer so as to extend the primer); introducing said cleave reagent under conditions so as to remove said cleavable protecting group (e.g. by cleving it) and (optionally) said detectable label from said modified primer/template hybridization complex (e.g. by cleving the linker)
Implementation Method 2
Moreover, the present invention provides embodiments where the thiol-containing compounds are scavenged with an oxidative wash step
Data Source
AI summary
The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. In addition, thiol-containing compounds and scavengers of thio-containing compounds are described. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.