Large-Stokes-Shift Fluorescent Dyes for Multiplex PCR Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current fluorescence-based PCR technologies are limited in their ability to analyze multiple targets in a single reaction vessel due to spectral overlap of fluorophores, allowing for the distinction of only four to six individual PCR targets using optical filters.
Innovation Solution
Development of fluorescent dyes with a large Stokes shift (>50 nm) and thermostable fluorescence, incorporating specific linker moieties that facilitate tuning of spectroscopic properties and enable conversion into activated derivatives for bio-molecular labeling, accessible from inexpensive starting materials in a single reaction step.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard fluorophores are used with optical filters to detect multiple targets, then the detection system can identify up to six individual PCR targets, but spectral overlap between fluorophores limits the number of distinguishable targets
Solution Approach 1:
The patent changes the spectral parameters of the fluorophores by using dyes with large Stokes shifts (greater than 50 nm, preferably greater than 100 nm). This parameter change separates the excitation and emission wavelengths more significantly, reducing spectral overlap and enabling detection of more than six targets in a single reaction vessel while maintaining measurement precision through reduced interference between channels.
2Quantity of substance
If dyes with large Stokes shift are used to increase multiplexing capability, then more targets can be detected in a single tube, but the complexity of dye selection and optimization increases
Solution Approach 1:
The patent develops a universal approach to creating dyes with large Stokes shifts by using a common synthetic method that applies to multiple dye structures. The general procedure involves reacting a cyanine dye with a specific linker molecule under standardized conditions, which can be applied across different dye variants. This universal method reduces the complexity of dye selection and optimization by providing a systematic framework rather than requiring individual optimization for each dye.
Solution Approach 2:
The patent systematically varies specific parameters (such as the R1 and R2 groups in the dye structure) while maintaining the core synthetic methodology. This parameter-based approach allows exploration of multiple dye options with different spectral properties without redesigning the entire synthesis process, thereby reducing overall complexity while enabling detection of more targets.
3Productivity
If fluorescent dyes are used for real-time PCR detection, then simultaneous amplification and detection can be achieved, but spectral overlap of fluorophores limits the number of multiplexed targets
Solution Approach 1:
The patent changes the spectral parameters of the fluorophores by using dyes with large Stokes shifts (greater than 50 nm, preferably greater than 100 nm). This parameter change separates the excitation and emission wavelengths more significantly, reducing spectral overlap and enabling detection of more than six targets in a single reaction vessel while maintaining measurement precision through reduced interference between channels.
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
Enables the differentiation of multiple targets in a single reaction vessel by reducing spectral overlap, allowing for enhanced multiplexing capabilities in fluorescence-based nucleic acid detection technologies.
Implementation Method 1
The probe is preferably labeled with one or more fluorescent moieties, which absorb and emit light at specific wavelengths. Upon hybridizing to the target sequence or its amplicon, the probe exhibits a detectable change in fluorescent emission
Implementation Method 2
The present disclosure is directed to dyes having a large Stokes shift, such as a Stokes shift of about 50 nm or more, of about 60 nm or more, of about 70 nm or more, of about 80 nm or more, of about 90 nm or more, etc.)
Implementation Method 3
the incorporation of specific linker moieties into the dyes of the present disclosure allow for facile tuning of their spectroscopic properties... by choosing suitable linker moieties the dyes of the present disclosure may be converted to their respective activated derivatives
Data Source
AI summary
The present disclosure relates, in general, to novel and easily accessible fluorescent compounds with large Stokes shift (LSS) and thermostable fluorescence for expanding the multiplexing capabilities of fluorescence-based nucleic acid detection technologies. Moreover, conjugates, probes and FRET pairs comprising these fluorescent compounds as well as methods for amplification and detection of a target nucleic acid utilizing these fluorescent compounds and methods of labeling are also provided.