Methylation-Specific Quantitative Melt Analysis for Fragile X Syndrome
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Solution Overview
Problem
Current diagnostic assays for fragile X syndrome and related disorders lack accuracy in quantifying methylation levels, particularly in females and for predicting disease severity, due to limitations in sensitivity and throughput, and require specialized equipment and expertise.
Innovation Solution
A methylation-specific quantitative melt analysis (MS-QMA) method that involves bisulfite treatment, real-time PCR, and high-resolution melt analysis to quantify methylation levels at specific CpG sites, using a standard curve to determine percentage methylation, which can be performed in standard diagnostic laboratories without specialized equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If methylation sensitive southern blot is used for molecular diagnosis of FXS, then information on CGG size and methylation status is obtained, but the assay is time consuming and low throughput
Solution Approach 1:
The patent replaces the mechanical blotting and hybridization steps of southern blot with a PCR-based amplification system that uses methylation-sensitive restriction enzymes and fluorescently labeled probes. This substitution enables automated high-throughput processing while maintaining the ability to detect both CGG expansion size and methylation status simultaneously.
Solution Approach 2:
The patent changes the detection parameters by using fluorescently labeled oligonucleotide probes that hybridize to specific regions within the FMR1 gene. By monitoring fluorescence signals during real-time PCR, the system can quantify methylation levels and determine CGG repeat size in a high-throughput format, transforming the low-throughput southern blot into a quantitative real-time assay.
2Productivity
If PCR based diagnostic assays targeting only CGG expansion size are used, then the test is simpler and faster, but the results cannot provide accurate prognostic information on disease type and severity
Solution Approach 1:
The patent merges two separate diagnostic functions into a single PCR assay: (1) detection of CGG expansion size through amplification of the repeat region, and (2) determination of methylation status through incorporation of methylation-sensitive restriction enzyme digestion and fluorescent probes. This combination allows the assay to provide both diagnostic speed and prognostic accuracy simultaneously.
Solution Approach 2:
The patent introduces fluorescently labeled oligonucleotide probes as intermediaries that bind to specific methylation-sensitive sites within the FMR1 gene. These probes serve as mediators that translate methylation status into quantifiable fluorescent signals, enabling the assay to provide accurate prognostic information while maintaining high throughput.
3Device complexity
If methylation sensitive PCR is used for diagnosis, then the assay is simpler than southern blot, but it is not highly sensitive or quantitative and cannot detect methylation mosaicism accurately
Solution Approach 1:
The patent replaces the qualitative endpoint detection of traditional methylation-sensitive PCR with real-time fluorescent detection during the PCR amplification process. This substitution enables quantitative measurement of methylation levels by monitoring fluorescence signals that correlate with the amount of amplified product, thereby achieving both simplicity and quantitative accuracy.
Solution Approach 2:
The patent incorporates real-time fluorescence monitoring that provides continuous feedback during the PCR amplification process. By measuring fluorescence at each cycle, the system can accurately quantify methylation levels and detect mosaicism, as the fluorescent signal intensity directly reflects the amount of methylated versus unmethylated DNA being amplified.
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 accurate, high-throughput quantitation of methylation levels, providing prognostic value and correlating with clinical phenotypes, particularly in young children, and can be used for various neurological and developmental disorders, including fragile X syndrome, without the need for expensive equipment or advanced training.
Implementation Method 1
subjecting the DNA sample to bisulfite treatment to convert non-methylated cytosine nucleotides in the DNA to uracil nucleotides
Implementation Method 2
amplifying a portion of the DNA sample comprising the target region
Implementation Method 3
melting the amplified DNA and analyzing the melt curve to determine methylation status
Data Source
Figure 1A~1G
Figure 1H
Figure 2(i)
AI summary
The present specification relates to an assay to quantitate extent of methylation in a DNA sample. Kits and clinical diagnostic assays are also enabled herein including assays to determine clinical phenotypes based on extent of methylation of a DNA target site.