Nucleotide-Specific Ternary Complex Detection for Sequencing Accuracy
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in achieving extended sequencing read-length with improved base-calling accuracy across different platforms, necessitating a method for precise determination of the next correct nucleotide in a template strand.
Innovation Solution
A method involving contacting a primed template nucleic acid with reaction mixtures containing DNA polymerase and test nucleotides, measuring binding interactions without chemical incorporation, and determining the correct nucleotide based on measured binding results, which can be repeated multiple times to enhance sequence accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent Sanger dideoxyribonucleotide sequencing techniques are used, then sequence determination can be accomplished, but sequencing read-length is limited and base-calling accuracy cannot be improved across different platforms
Solution Approach 1:
The sequencing process is divided into distinct phases: binary complex formation (polymerase binding to primed template), ternary complex formation (addition of correct nucleotide), and detection. This segmentation allows for enhanced measurement precision by focusing detection on the specific ternary complex formation event, enabling improved base-calling accuracy without increasing overall platform complexity
Solution Approach 2:
The patent introduces an intermediary detection mechanism that measures binding interactions between the polymerase and the primed template nucleic acid in the presence of test nucleotides. This intermediary measurement approach enables accurate determination of the next correct nucleotide without requiring complex fluorescent labeling systems, thereby improving base-calling accuracy while maintaining platform simplicity
2Productivity
If sequencing by hybridization or sequencing-by-synthesis is used, then automated processing can be achieved, but extended sequencing read-length with improved base-calling accuracy remains challenging
Solution Approach 1:
The method incorporates feedback through iterative cycles of nucleotide testing and binding measurement. By repeatedly contacting the primed template with different test nucleotides and measuring binding results, the system accumulates information to determine the correct nucleotide identity with high confidence. This feedback mechanism enables both automated processing and improved base-calling accuracy
Solution Approach 2:
The patent performs preliminary binding measurements to assess polymerase-nucleotide complex formation before committing to nucleotide incorporation. This preliminary action allows the system to accurately identify the correct nucleotide through binding affinity measurement, ensuring high base-calling accuracy while maintaining automated processing efficiency through standardized protocol steps
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 accurate and extended nucleic acid sequencing with improved base-calling accuracy by distinguishing specific ternary complex formation from nonspecific binary complexes, allowing for controlled reaction conditions and unambiguous sequence determination.
Implementation Method 1
distinguishing specific ternary complex formation from nonspecific binary complexes
Implementation Method 2
measuring binding interactions without chemical incorporation
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. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.