Cognate Nucleotide Detection Through Nonincorporating Ternary Complexes
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Solution Overview
Problem
Current next-generation sequencing (NGS) methods using labeled reversible terminators are slow and inefficient, leading to undesired phasing and base calling errors due to repetitive chemistry steps and incomplete removal of reversible terminator moieties.
Innovation Solution
A method involving a blocked primed template nucleic acid molecule immobilized on a solid support, contacted with a polymerase and a nucleotide analog that forms a ternary complex without nucleotide incorporation, allowing for the detection of the next correct nucleotide using a detectable label.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeled reversible terminator nucleotides are used for stepwise identification, then nucleotide identification can be achieved, but the sequencing workflow becomes slow and inefficient
Solution Approach 1:
The patent extracts and removes the reversible terminator moiety from the nucleotide structure, using only the detectable label-containing nucleotide analog without the blocking group. This eliminates the need for repetitive incorporation and removal cycles, enabling direct detection of the next correct nucleotide and dramatically improving sequencing speed while maintaining identification accuracy.
Solution Approach 2:
Instead of incorporating a nucleotide and then removing the terminator to proceed to the next step (conventional approach), the patent inverts the logic by using a nucleotide analog that forms a stable ternary complex with the polymerase and template without incorporation. This allows direct detection of the nucleotide identity in the complex, reversing the traditional sequence of operations to achieve faster sequencing.
2Measurement precision
If repetitive chemistry steps are used for nucleotide identification, then nucleotide can be identified, but phasing and base calling errors are introduced
Solution Approach 1:
The patent removes the reversible terminator chemistry from the process entirely, eliminating the complex repetitive steps of incorporation, labeling, and removal. By using a nucleotide analog that forms a stable ternary complex without requiring incorporation, the chemistry is simplified to a single detection step, reducing phasing errors and base calling inaccuracies associated with multiple repetitive cycles.
Solution Approach 2:
The patent introduces a stabilizing agent as an intermediary that promotes formation of a stable ternary complex between the polymerase, template, and nucleotide analog. This intermediary enables the complex to remain stable long enough for detection without requiring the nucleotide to be incorporated into the DNA strand, simplifying the chemistry and improving accuracy by eliminating repetitive steps.
3Productivity
If reversible terminator moiety removal is performed on only some blocked primed template nucleic acids, then the next round of incorporation can proceed, but correct nucleotide identification loses register
Solution Approach 1:
The patent extracts and eliminates the reversible terminator moiety from the nucleotide structure before the detection step. By using a nucleotide analog without the blocking group that forms a stable ternary complex, there is no need for subsequent removal steps. This ensures that all nucleic acid molecules are in the same state (deblocked) and ready for uniform detection, preventing register loss that occurs when only some molecules have their terminators removed.
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 enables rapid and efficient nucleotide identification, reducing errors and improving sequencing speed by eliminating the need for repetitive chemistry steps and ensuring complete removal of reversible terminators.
Implementation Method 1
contacting the blocked primed template nucleic acid molecule with (i) a polymerase, and (ii) a nucleotide analog, whereby a ternary complex forms, without nucleotide incorporation
Implementation Method 2
detecting the detectable label of the nucleotide analog present in the ternary complex isolated in step (c)
Data Source
AI summary
Method and composition for identifying cognate nucleotides in a Sequencing By Binding™ procedure, wherein one or more labeled nucleotides are detected in ternary complexes but never incorporated. Labeled nucleotides can be incorporable nucleotides that contact preformed blocked primed template nucleic acids. Alternatively, labeled nucleotides are labeled non-incorporable nucleotides. Labeled nucleotides, including labeled non-incorporable nucleotides, can be detected in ternary complexes in the same reaction mixture that incorporates a reversible terminator nucleotide to create a blocked primed template nucleic acid. Detection of ternary complexes can take place in the presence of a catalytic metal ion.