Sequencing Additive for DNA Template Protection
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Solution Overview
Problem
Current DNA sequencing technologies, particularly Sequencing-by-Synthesis (SBS), face challenges in achieving longer read lengths and higher accuracy, with existing methods lacking effective solutions for deprotection conditions and enzymatic incorporation of nucleoside triphosphates with nitrate groups and 3'-OH-blocking agents.
Innovation Solution
The use of 5-Chloro-2-methyl-4-isothiazolin-3-one and thiol-containing compounds as cleaving agents for disulfide-based linkers and 3'-disulfide capping groups in deoxynucleoside triphosphates, enabling reversible protection and detection of nucleotides, and the incorporation of nucleotide analogues with methylenedisulfide as a cleavable protecting group and a detectable label attached via a cleavable disulfide linker.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If disulfide-based 3'-terminator groups are used for reversible blocking in SBS, then deprotection can be achieved by reducing agents, but the process causes DNA template damage and limits read length
Solution Approach 1:
The patent changes the chemical nature of the blocking group from disulfide to methyl phosphonate ester, which fundamentally alters the deprotection mechanism from reduction to hydrolysis, thereby eliminating DNA template damage while maintaining reversible blocking functionality
Solution Approach 2:
The blocking group is designed as a temporary, disposable protective element that is easily removed without affecting the underlying DNA template, allowing the template to be reused for multiple sequencing cycles without degradation
2Ease of manufacture
If nitrate groups are used as 3'-OH-blocking agents, then deprotection conditions can be simplified, but enzymatic incorporation efficiency is reduced
Solution Approach 1:
The patent modifies the blocking group chemistry from nitrate to methyl phosphonate ester, optimizing the balance between enzymatic acceptance and deprotection ease by creating a group that polymerases can efficiently incorporate while still allowing simple base-catalyzed hydrolysis for removal
3Length of stationary object
If longer read lengths are pursued in SBS, then more nucleotides can be sequenced, but accuracy decreases due to cumulative errors
Solution Approach 1:
The patent extracts the source of cumulative errors by eliminating DNA template damage caused by traditional deprotection methods, thereby maintaining template integrity throughout extended sequencing cycles and preserving accuracy over longer read lengths
Solution Approach 2:
The patent implements preliminary protection of the DNA template by using blocking groups that can be removed without damaging the template, ensuring the template remains intact and reusable for multiple sequential nucleotide incorporations
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 the accuracy and length of DNA sequencing by facilitating efficient incorporation and detection of nucleotides, improving the read length and reducing DNA template damage, while maintaining sequencing performance and reducing lead in sequencing reactions.
Implementation Method 1
utilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencing reactions, and in particular, sequencing reactions where deoxynucleoside triphosphates comprising a 3'-O position capped by a disulfide-based 3'-terminator group are used
Implementation Method 2
The present invention also provides methods, compositions, mixtures and kits utilizing thiol-containing compounds or derivatives (alternatively mercaptol analogues) as cleave agents of the disulfide based linkers and 3'-disulfide capping group of nucleotides
Data Source
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AI summary
The present-invention provides methods, compositions, mixtures and kits utilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencing reactions, and in particular, sequencing reactions where deoxynucleoside triphosphates comprising a 3'-O position capped by a disulfide-based 3'-terminator group are used. In one embodiment, the deoxynucleoside triphosphates comprise 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.