Sequencing Method Using Ternary Complex Destabilization
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in accurately determining sequences, particularly with homopolymer stretches, which can lead to ambiguous results due to phasing issues and increased reagent costs from iterative cycles.
Innovation Solution
A method involving a primed template nucleic acid with a reversible terminator moiety, where a ternary complex is formed with a polymerase and multiple nucleotides, and then investigated under changing reagent conditions to identify the next base by monitoring the stability of the complex, reducing the number of steps and reagents needed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If iterative cycles of enzyme-based nucleotide binding and incorporation are used to identify cognate nucleotides, then sequencing accuracy can be improved, but workflow complexity and reagent costs increase
Solution Approach 1:
The patent extracts the nucleotide identity determination step from the iterative sequencing cycle. By using a separate binding reaction that identifies which nucleotide binds to the template-primer complex without requiring incorporation, the method determines nucleotide identity in a single step rather than through multiple iterative cycles of binding, incorporation, and detection.
Solution Approach 2:
The patent segments the sequencing process into distinct functional steps: (1) nucleotide binding reaction to identify cognate nucleotide, (2) optional incorporation step, and (3) detection. This segmentation allows the binding and identity determination to occur independently from the incorporation cycle, reducing overall workflow complexity.
2Loss of information
If iterative cycles of nucleotide incorporation are performed to determine sequence, then complete sequence information can be obtained, but reagent costs increase
Solution Approach 1:
The patent performs preliminary nucleotide binding reactions to identify which nucleotide is cognate to each template position before performing incorporation. By determining nucleotide identity in advance through binding reactions with all four dNTPs present, the method avoids unnecessary incorporation reactions and reduces consumption of expensive labeled nucleotide reagents.
Solution Approach 2:
The patent uses unlabeled dNTPs as substitutes for expensive fluorescently labeled nucleotides in the binding determination step. By performing identity determination with cheap unlabeled nucleotides and only using labeled nucleotides when incorporation is actually needed, the method significantly reduces reagent costs while maintaining complete sequence information.
3Measurement precision
If homopolymer stretches are present in the template, then sequencing accuracy decreases due to phasing issues, but the template sequence remains unchanged
Solution Approach 1:
The patent introduces a binding reaction as an intermediary step between template presentation and nucleotide incorporation. By allowing all four dNTPs to compete for binding to the template-primer complex in a controlled binding reaction, the method ensures that only the correct cognate nucleotide binds stably, even in homopolymer regions. This intermediary binding step prevents phasing errors by ensuring accurate nucleotide identification before incorporation.
Solution Approach 2:
The patent uses dynamic binding conditions where all four dNTPs are present in the binding reaction, allowing equilibrium binding to occur. The stable binding of the correct nucleotide in homopolymer regions can be detected through this dynamic process, preventing the phasing issues that occur when incorrect nucleotides are incorporated due to homogeneous sequences.
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 technique enables clear and unambiguous detection of nucleotides, reduces reagent costs, and simplifies the sequencing process by allowing single incorporation reactions to determine multiple positions along a nucleic acid strand.
Implementation Method 1
contacting the blocked primed template nucleic acid molecule with a first reaction mixture that includes a polymerase, and a plurality of different nucleotide molecules, whereby a stabilized ternary complex forms, the stabilized ternary complex including one of the plurality of different nucleotide molecules
Implementation Method 2
monitoring interaction of the polymerase and the blocked primed template nucleic acid molecule in contact with the second reaction mixture to detect any of the stabilized ternary complex remaining after step (c)
Data Source
AI summary
Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.