PCR Colorant Segmentation for Pipetting Accuracy
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Solution Overview
Problem
Current pipetting aids for PCR are not effective in the sample pipetting phase and are not compatible with quantitative PCR, as they interfere with real-time optical measurements due to overlapping spectral properties, and mechanical solutions are expensive and prone to errors.
Innovation Solution
The use of two reagent solutions colored with different initial colorants that produce a distinct third color upon mixing, allowing visual verification of the pipetting process without interfering with qPCR fluorescence, and the inclusion of additional colorants to create further distinguishable colors for different stages of the PCR process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If colorants are added to PCR master mixes to aid pipetting visualization, then pipetting accuracy is improved, but the colorants interfere with real-time optical measurements in quantitative PCR
Solution Approach 1:
The patent divides the colorant function into two separate components: a first colorant added to the master mix and a second colorant added to the sample. This segmentation allows each colorant to be optimized independently - the first colorant provides visualization aid for pipetting while the second colorant ensures compatibility with qPCR detection wavelengths, thereby resolving the contradiction between pipetting accuracy and optical measurement accuracy
Solution Approach 2:
The patent changes the spectral parameters of the colorants by selecting specific dyes with non-overlapping absorption spectra. The first colorant (e.g., bromophenol blue) absorbs in the visible range while the second colorant (e.g., xylene cyanol) absorbs at different wavelengths, ensuring that neither interferes with the fluorescence detection wavelengths used in qPCR. This parameter optimization resolves the contradiction by maintaining both visualization capability and optical measurement integrity
2Manufacturing precision
If mechanical automated pipetting systems are used to improve pipetting accuracy, then pipetting precision is improved, but the system complexity and cost increase significantly
Solution Approach 1:
The patent implements a self-service approach where the reaction mixture itself provides visual feedback about pipetting accuracy through color changes. The colored master mix and colored sample produce a visibly distinct mixed solution, allowing operators to immediately verify correct pipetting without requiring complex automated systems. This simple visual self-verification mechanism achieves high pipetting precision while avoiding the complexity and cost of mechanical automated systems
3Reliability
If density enhancers are added to PCR mixes to improve gel loading, then electrophoresis performance is improved, but the solution viscosity and pipetting difficulty increase
Solution Approach 1:
The patent extracts the density enhancer function from the main reaction mixture by adding it separately as a distinct component. This allows the density enhancer to be optimized for gel loading performance without necessarily compromising the pipetting characteristics of the main mixture. The separated addition enables independent optimization of both gel loading reliability and pipetting ease
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 method enhances pipetting accuracy by providing a visual means to verify proper mixing and reaction composition, reduces errors, and is compatible with qPCR by minimizing absorbance at fluorescence wavelengths, thus maintaining the integrity of optical detection.
Implementation Method 1
mixing the first and second reagent solutions for providing a mixed solution to be subjected to the PCR process, the mixed solution having, due to said first and second colorants, a third color different from the first and second colors
Implementation Method 2
the absorbance peaks of any of said colorants do not overlap with the emission or excitation wavelength of said fluorescent dye, primer or probe
Data Source
Figure 1A~1B
Figure 2a
Figure 2b
AI summary
The invention relates to a method of preparing a reaction mixture for Polymerase Chain Reaction (PCR)assay and a solution set for PCR. The method comprises providing a sample solution comprising a biological sample to be amplified in said PCR assay and first colorant providing the solution a first color, providing a reagent solution comprising at least one other substance required for performing said assay and second colorant providing the solution a second color different from the first color, and mixing the sample solution and the first reagent solution for providing a mixed solution to be subjected to the PCR process, the mixed solution having, due to said first and second colorants, a third color different from the first and second colors. The invention significantly aids in pipetting PCR assays.