Reversible Terminator with Cleavable Linker for DNA Sequencing
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Solution Overview
Problem
High-throughput nucleic acid sequencing technologies face challenges in achieving accurate and efficient genome information delivery, often compromising on accuracy for speed and cost, particularly in sequencing by synthesis methodologies where reversible terminator molecules leave behind residual linker structures that impede further polymerase activity.
Innovation Solution
Development of reversible terminator molecules with a 3′-azidoalkanoate blocking group on the 3′-O of the ribose ring, recognized by DNA polymerase for incorporation, followed by click chemistry with a terminal alkyne label for detection and subsequent mild cleavage to regenerate the 3′-OH for continuous elongation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reversible terminator molecules with residual linker structures are used for high-throughput sequencing, then sequencing speed and cost efficiency are improved, but polymerase activity is impeded and sequencing accuracy deteriorates
Solution Approach 1:
The patent extracts and removes the harmful residual linker structures from the reversible terminator molecules through specific chemical cleavage methods. By designing terminators with cleavable linkers that can be removed after the blocking function is no longer needed, the patent eliminates the impediment to polymerase activity while maintaining the high-throughput sequencing capability.
Solution Approach 2:
The patent changes the chemical parameters of the reversible terminator molecules by introducing specific functional groups and cleavable linkers. These parameter changes allow the terminators to be removed under controlled conditions (e.g., basic treatment, enzymatic cleavage), thereby restoring polymerase activity and improving sequencing accuracy without sacrificing productivity.
2Measurement precision
If reversible terminator molecules are used to determine incorporated nucleotides, then measurement precision is improved, but residual structures hinder further polymerase activity
Solution Approach 1:
The patent applies preliminary action by designing the reversible terminator with a built-in cleavable linker that is prepared for removal after serving its dual function of blocking and labeling. This preliminary design ensures that after nucleotide identification, the terminator can be easily removed to restore polymerase activity, thus maintaining ease of operation throughout the sequencing process.
Solution Approach 2:
The cleavable linker acts as an intermediary that temporarily connects the blocking group to the nucleotide. After the blocking function serves its purpose for accurate nucleotide identification, the intermediary linker can be selectively cleaved, allowing the blocking group to be removed without damaging the incorporated nucleotide or the DNA strand, thus restoring polymerase activity.
3Productivity
If standard reversible terminators are used in sequencing by synthesis, then high-throughput capability is achieved, but residual linker structures accumulate and impede continuous elongation
Solution Approach 1:
The patent implements discarding and recovering by designing the reversible terminator to be discarded (removed) after serving its function. The cleavable linker enables selective removal of the terminator while recovering the 3'-OH group, allowing continuous elongation. This process can be repeated for multiple sequencing cycles, maintaining high throughput while achieving longer read lengths.
Solution Approach 2:
The patent ensures continuity of useful action by designing the terminator removal process to be efficient and complete. By using cleavable linkers that can be removed under mild conditions, the patent maintains continuous polymerase activity across multiple sequencing cycles, enabling longer read lengths without interruption from accumulated residual structures.
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 accurate determination of incorporated nucleotides without damaging the DNA strand or polymerase, enabling longer read lengths by preventing residual linker structures from hindering further polymerase activity, thus enhancing sequencing efficiency and accuracy.
Implementation Method 1
The cleavable linker comprises a disulfide bond which can be cleaved by a reducing reagent
Implementation Method 2
the covalent linkage to the 3′ hydroxyl is reversible, meaning the cleavable chemical group may be removed by chemical and/or enzymatic processes
Implementation Method 3
the covalent linkage to the 3′ hydroxyl is reversible, meaning the cleavable chemical group may be removed by chemical and/or enzymatic processes
Implementation Method 4
recognized by DNA polymerase for incorporation
Implementation Method 5
click chemistry with a terminal alkyne label for detection
Data Source
AI summary
The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3′OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3′-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V):