Genotypic Analysis Spectral Calibration via Fluorescence Shift Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional spectral calibration methods require separate electrophoresis of a matrix standard before analyzing actual samples, which is time-consuming and costly, and necessitates the use of consumable standards.
Innovation Solution
A method for genotypic analysis that performs spectral calibration simultaneously with the analysis of actual samples using a size standard and allelic ladder, detecting shifts in fluorescence spectra to determine reference spectra without the need for a special matrix standard.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate electrophoresis of matrix standard is performed before sample analysis, then spectral calibration accuracy is improved, but analysis time increases and cost increases
Solution Approach 1:
The patent combines the spectral calibration process with the actual sample analysis process by using the size standard and allelic ladder that are already present in the sample mixture. Instead of performing separate electrophoresis of a matrix standard, the method utilizes the fluorescently labeled DNA fragments from the size standard to obtain reference fluorescence spectra simultaneously with sample analysis, thereby eliminating the need for additional calibration time while maintaining calibration accuracy.
Solution Approach 2:
The size standard components in the sample mixture serve dual purposes: they function as both the analytical sample and the calibration reference. The fluorescently labeled DNA fragments of known sizes from the size standard automatically provide the reference fluorescence spectra needed for calibration, making the system self-calibrating without requiring external matrix standard materials or separate calibration procedures.
2Measurement precision
If separate electrophoresis of matrix standard is performed, then spectral calibration is achieved, but consumable cost increases
Solution Approach 1:
The size standard, which is already included in the sample analysis mixture, is made to serve multiple functions: it acts as both the analytical reference for determining unknown DNA fragment sizes and as the calibration standard for obtaining reference fluorescence spectra. This multi-functionality eliminates the need for separate matrix standard consumables, reducing material costs while maintaining calibration capability.
Solution Approach 2:
The system uses its own inherent size standard components to perform spectral calibration, making the calibration process self-sufficient. The fluorescently labeled DNA fragments from the size standard automatically provide both the analytical reference data and the calibration reference spectra, eliminating the need for additional consumable matrix standards and reducing overall operational costs.
3Measurement precision
If fluorescence spectra are measured for each capillary in capillary array, then spectral calibration accuracy is improved, but measurement time and complexity increase
Solution Approach 1:
The patent merges the spectral calibration measurements for multiple capillaries into a single simultaneous electrophoresis run. By using the size standard present in all capillaries and detecting its fluorescence spectra across all capillaries at once, the method obtains reference spectra for each capillary without requiring separate measurement procedures, thereby simplifying the calibration process while maintaining per-capillary calibration accuracy.
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 allows for rapid and cost-effective spectral calibration by eliminating the need for separate matrix standard electrophoresis, reducing the time and expense associated with calibration.
Implementation Method 1
Excitation light of a fluorescent dye obtained by irradiating a capillary end with laser light of a specific wavelength is separated in the wavelength direction with a diffraction grating
Implementation Method 2
Electrophoresis is a method for separating DNA fragments using the phenomenon that the migration speed in a charged migration path varies with the DNA fragment length
Implementation Method 3
Each sample, namely each DNA fragment, is labeled with a fluorescent dye, and the fluorescence signal from the migrated sample is detected with an optical detector
Data Source
Figure 1
Figure 2
Figure 3~4A
AI summary
A technique for performing spectral calibration simultaneously with electrophoresis of an actual sample to be analyzed, without performing electrophoresis using a special matrix standard which is time-consuming and costly, is provided. The device for genotypic analysis is characterized by obtaining reference fluorescence spectra using a size standard and an allelic ladder, which provide information concerning known DNA fragments used for electrophoresis of an actual sample, and is characterized by performing spectral calibration for a capillary in which the allelic ladder is not used by detecting a shift amount of the fluorescence spectra of the size standard and shifting the reference fluorescence spectra using the shift amount to determine fluorescence spectra.