Rhodamine Dye Compounds for Multiplex Nucleic Acid Sequencing
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Solution Overview
Problem
Current fluorescent dye compounds for nucleic acid detection face challenges in multiplexing due to spectral overlap and compatibility issues with reagents, limiting their use in high-throughput molecular biology methods like solid phase sequencing.
Innovation Solution
Development of novel rhodamine dye compounds with specific structural formulas and linker groups for conjugation to nucleosides and nucleotides, enabling efficient fluorescence detection and sequencing by synthesis while being compatible with DNA synthesis reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent dyes are used for multiplex detection, then the detection capability for multiple analytes is improved, but spectral overlap and compatibility issues with reagents worsen
Solution Approach 1:
The patent modifies the chemical structure of fluorescent dyes by introducing specific substituent groups (R1-R9) and counterions (M+), which changes their spectral properties. This allows optimization of emission wavelengths to achieve better spectral separation while maintaining compatibility with DNA synthesis reagents, thereby resolving the contradiction between multiplex capability and spectral overlap
Solution Approach 2:
The patent applies different substituent groups at specific positions on the dye molecule structure (R1 at position 1, R2 at position 2, etc.), creating dyes with localized functional properties. This enables each dye to have optimized spectral characteristics for specific detection wavelengths, improving multiplex detection while reducing spectral overlap between different dye channels
2Power
If high power lasers are used for excitation, then the excitation efficiency is improved, but photo-stability requirements become more stringent
Solution Approach 1:
The patent optimizes the dye molecular structure with specific substituent groups and counterions to enhance photo-stability parameters. This allows the dyes to withstand high power laser excitation without degrading, thereby enabling efficient excitation while maintaining reliability in automated sequencing instruments
3Measurement precision
If fluorescent dyes are selected for spectral resolution, then the detection specificity is improved, but compatibility with DNA synthesis reagents may worsen
Solution Approach 1:
The patent carefully selects counterions (M+) and substituent groups (R1-R9) to optimize both spectral properties and chemical compatibility. The sulfonate or sulfonic acid groups provide water solubility and compatibility with aqueous DNA synthesis buffers, while the aromatic substituents tune the spectral characteristics, thereby achieving both detection specificity and reagent compatibility simultaneously
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 novel rhodamine dye compounds provide spectrally distinguishable labels for multiplex detection, enhancing the throughput and accuracy of nucleic acid sequencing and analysis, and are compatible with DNA synthesis reagents, addressing the limitations of existing dyes.
Implementation Method 1
Non-radioactive detection of nucleic acids utilizing fluorescent labels
Data Source
AI summary
Novel rhodamine dye compounds, labelled conjugates comprising the dyes are described, together with methods for their use. The dyes and labelled conjugates are useful as molecular probes in a variety of applications, such as in assays involving staining of cells, protein binding, and analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.