Protected Dye-Labeled Nucleotide Reagents for Low-Photodamage Sequencing
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Solution Overview
Problem
Next-generation sequencing technologies face challenges in achieving higher throughput at lower cost per base sequenced, with issues related to fidelity, processivity, and accuracy impacting the quality of sequencing data, particularly in enzyme-mediated, template-dependent sequencing processes.
Innovation Solution
Development of dye-labeled compounds comprising donor and acceptor dyes, shield elements, and terminal coupling elements, which modulate the kinetics of polymerase reactions to improve sequencing performance, including the use of avidin protein shields to associate with nucleotide and dye-labeled compounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If higher throughput sequencing is implemented, then productivity increases, but measurement precision and reliability deteriorate
Solution Approach 1:
The patent modifies the chemical and physical parameters of nucleotide reagents by incorporating fluorescent labels at specific positions (such as the 2'-OH group of the ribose ring) and adjusting kinetic properties through molecular design. These parameter changes enable real-time detection of incorporation events while maintaining sequencing accuracy, resolving the contradiction between throughput and measurement precision.
Solution Approach 2:
The patent replaces traditional mechanical or chemical detection methods with fluorescence-based optical detection. By using fluorescently labeled nucleotides that emit light upon incorporation, the system achieves real-time, non-invasive detection that maintains accuracy while enabling higher throughput sequencing.
2Measurement precision
If fluorescent labels are incorporated into nucleotide reagents, then measurement precision improves, but object-generated harmful factors increase due to photodamage
Solution Approach 1:
The patent employs nucleotide analogs with fluorescent labels that are designed to be incorporated transiently and then removed or degraded after serving their detection purpose. This disposable approach allows for real-time detection without long-term photodamage accumulation, as the fluorescent labels are not permanently retained in the final sequenced product.
Solution Approach 2:
The patent extracts or removes the fluorescent label from the nucleotide after the incorporation event has been detected. By separating the detection function (fluorescent label) from the final sequencing product, the system maintains measurement precision while eliminating the source of photodamage for subsequent cycles.
3Productivity
If modified nucleotide reagents with improved kinetic properties are developed, then productivity increases, but device complexity increases
Solution Approach 1:
The patent segments the nucleotide reagent into distinct functional components: the nucleotide core, the fluorescent label, and kinetic modification groups. This segmentation allows each component to be optimized independently for its specific function while maintaining overall compatibility, enabling improved sequencing speed without proportionally increasing overall complexity.
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
The improved nucleotide analogs reduce photodamage and enhance brightness, leading to shortened interpulse distances and increased signal-to-noise ratios, thereby enhancing the accuracy and efficiency of sequencing reactions.
Implementation Method 1
a donor dye; an acceptor dye
Data Source
AI summary
Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.