Large Stokes Shift Fluorescent Dyes for Multiplexed PCR
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Solution Overview
Problem
Current real-time PCR methods are limited in their ability to simultaneously detect and quantify multiple nucleic acid targets due to the constraints of spectrally resolvable fluorophores, allowing for detection of only four to seven separate targets in a single reaction vessel, which is insufficient for clinical needs.
Innovation Solution
The use of large Stokes shift (LSS) fluorescent dyes in combination with oligonucleotide probes and nucleic acid polymerases having 5′ to 3′ nuclease activity allows for the detection of multiple targets by minimizing spectral overlap and enabling the expansion of multiplexing capabilities without requiring changes to existing PCR instruments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorescent probes are used in the same reaction to detect multiple targets, then the number of detectable targets increases, but the spectral overlap between probes increases causing detection interference
Solution Approach 1:
The patent changes the fundamental parameter of fluorescent dye selection by using Large Stokes Shift (LSS) dyes instead of conventional dyes. LSS dyes have a large difference between absorption and emission wavelengths, which allows for greater spectral separation between multiple probes. This parameter change enables detection of more than 4-7 targets in a single reaction vessel by minimizing spectral overlap interference through optimized excitation and emission wavelength selection.
2Adaptability or versatility
If conventional fluorescent dyes are used with limited spectral resolution, then the detection system remains simple, but the multiplexing capacity is limited to 4-7 targets
Solution Approach 1:
The patent changes the optical parameters of the fluorescent dyes by switching to LSS dyes with large Stokes shifts. This allows the existing detection system to achieve higher multiplexing capacity (up to 21 targets) without requiring complex spectral discrimination capabilities, as the large wavelength separation between probes naturally reduces spectral overlap and interference.
3Loss of information
If more fluorescent probes are added to detect additional targets, then the information about multiple targets is improved, but the signal interference between probes increases
Solution Approach 1:
The patent changes the emission wavelength parameter of the fluorescent probes by using LSS dyes. This creates larger wavelength separations between the emission spectra of different probes, thereby reducing signal interference and allowing simultaneous detection of more targets (up to 21) with clearer, less overlapping signals that maintain high detectability.
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 the detection of up to 21 individual targets by creating additional optical channels, overcoming the limitations of standard fluorophores and allowing for both qualitative and quantitative analysis of multiple targets in a single reaction vessel.
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 use of large Stokes shift (LSS) fluorescent dyes in combination with oligonucleotide probes and nucleic acid polymerases having 5′ to 3′ nuclease activity allows for the detection of multiple targets by minimizing spectral overlap
Implementation Method 3
amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the 5′ to 3′ nuclease activity of the nucleic acid polymerase allows cleavage and separation of the LSS fluorescent dye from the first quenching moiety
Implementation Method 4
measuring the detectable signal from the LSS fluorescent dye by excitation at or near the wavelength of the absorption peak maximum of the LSS fluorescent dye
Data Source
AI summary
The present invention allows for the expansion of multiplexing capabilities of common PCR devices by using fluorogenic PCR probes made of large Stokes shift (LSS) fluorescent dyes. With this approach, no changes of the hardware or software components in the instrument are required.