Multinucleotide Repeat Detection via FRET Signal Ratio
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Solution Overview
Problem
Current methods for determining the length of multinucleotide repeat regions in nucleic acids, such as those associated with genetic disorders, are labor-intensive, time-consuming, and subjectively interpreted, often requiring costly and stringent capillary electrophoresis equipment.
Innovation Solution
The method involves amplifying target nucleic acids with a first label independent of repeat number and a second label proportional to the number of repeats, binding them to capture probes, and determining a signal ratio to assess the length of multinucleotide repeat regions, allowing for objective measurement without the need for size separation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Southern blot method is used to determine trinucleotide repeat length, then measurement capability is achieved, but labor intensity and time consumption increase significantly
Solution Approach 1:
The patent replaces the mechanical electrophoresis-based size separation system with a fluorescence resonance energy transfer (FRET) based optical detection system. By using labeled probes that hybridize to the repeat region and measuring FRET signals, the method eliminates the need for gel electrophoresis, thereby reducing labor intensity and time consumption while maintaining measurement capability.
Solution Approach 2:
The patent uses PCR amplification to create multiple copies of the target repeat region before detection. This allows a small amount of genomic DNA to be sufficient for analysis, speeding up the overall process by eliminating the need for large amounts of DNA and extensive sample preparation required by Southern blotting.
2Measurement precision
If electrophoresis-based methods are used, then size separation capability is achieved, but equipment cost and operational complexity increase
Solution Approach 1:
The patent substitutes complex electrophoresis equipment with a simple fluorescence detection system. The FRET-based assay requires only standard fluorescence readers or plate readers, which are widely available in clinical laboratories, thereby eliminating the need for expensive and maintenance-intensive capillary electrophoresis instruments.
Solution Approach 2:
The patent changes the detection parameter from physical size separation (electrophoresis mobility) to optical signal intensity (FRET efficiency). This parameter change allows repeat length determination through fluorescence signal ratios rather than migration distance, simplifying the equipment requirements while maintaining measurement precision.
3Productivity
If repeat primer PCR with electrophoresis is used, then amplification capability is achieved, but interpretation subjectivity increases
Solution Approach 1:
The patent replaces subjective visual interpretation of electrophoresis gels with objective fluorescence signal measurement. The FRET signal ratio provides a quantitative, numerical readout that can be automatically analyzed by software, eliminating inter-observer variability and subjectivity inherent in gel band interpretation.
Solution Approach 2:
The patent incorporates internal controls and reference signals within the FRET assay system that provide real-time feedback on assay performance. This includes using probes with known characteristics to validate each run, ensuring objective and reproducible results that can be automatically quality-controlled without subjective judgment.
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 provides a more efficient, objective, and cost-effective means to determine multinucleotide repeat lengths, reducing the reliance on expensive equipment and subjective interpretation, while accurately assessing the severity of genetic disorders.
Implementation Method 1
detecting the first label associated with the capture probe to produce a first signal; detecting the second label associated with the capture probe to produce a second signal
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
Methods of determining the length of a multinucleotide repeat region in a target nucleic acid are provided herein which include labeling amplified target nucleic acids with a target detection label independent of the number of multinucleotide repeats and a repeat-detection label proportional to the number of multinucleotide repeats, wherein the two types of labels are each independently incorporated in the amplified target nucleic acids during the amplifying or after the amplifying; binding the amplified target nucleic acids to a capture probe specific for the amplified target nucleic acids; detecting the target detection label associated with the capture probe to produce a first signal; detecting the repeat-detection label associated with the capture probe to produce a second signal; and determining a ratio of the first signal and the second signal, wherein the ratio is indicative of the length of the multinucleotide repeat region in the target nucleic acid.