Methylation Detection Probes for Partial DNA Analysis
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Solution Overview
Problem
Current methods for detecting DNA or RNA methylation, such as MethyLight, are limited in sensitivity due to requiring all targeted CpG sites to be methylated, leading to false negative results in cases of partial methylation, particularly in diagnostic applications like colorectal cancer detection.
Innovation Solution
A method involving primers and probes designed to amplify and detect both fully and partially methylated forms of DNA or RNA, using agents like sodium bisulfite to convert unmethylated cytosines, allowing for the detection of partial methylation patterns across CpG-rich regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methylation-specific PCR methods are used, then fully methylated DNA can be detected, but sensitivity is reduced and false negatives occur in cases of partial methylation
Solution Approach 1:
The patent segments the detection approach by using multiple probes with different methylation requirements. Some probes detect fully methylated DNA while others detect partially methylated DNA, allowing the system to capture all methylation states rather than relying on a single detection method that misses partial methylation cases
Solution Approach 2:
The patent changes the detection parameter by designing probes with varying degrees of methylation stringency. By adjusting probe design to match different methylation patterns (fully methylated, partially methylated, and unmethylated), the system can detect a broader range of methylation states, improving sensitivity without sacrificing reliability
2Measurement precision
If probes requiring all CpG sites to be methylated are used, then specific detection of fully methylated DNA is achieved, but detection of partially methylated DNA is lost
Solution Approach 1:
The patent creates a universal detection system where a single assay can detect multiple methylation states (fully methylated, partially methylated, and unmethylated DNA) using a panel of probes. This multi-functional approach allows the same PCR reaction to interrogate different methylation patterns, expanding the detection range while maintaining precision for each specific pattern
3Reliability
If high stringency probes are used for methylation detection, then false positives are reduced, but false negatives increase in partial methylation cases
Solution Approach 1:
The patent introduces dynamic probe selection where the stringency of probe binding can be adjusted based on the expected methylation state. By using a combination of high-stringency probes (for fully methylated detection) and lower-stringency probes (for partial methylation detection), the system dynamically adapts to detect different methylation levels while maintaining overall reliability through the panel approach
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 significantly enhances the sensitivity of methylation detection, reducing false negatives and improving diagnostic accuracy by enabling the detection of partially methylated DNA or RNA molecules.
Implementation Method 1
contacting a nucleic acid sample with an agent which modifies unmethylated cytosine residues
Data Source
Figure 1A~1B
Figure 2
Figure 2
AI summary
The present invention relates generally to a method for assessing nucleic acid methylation, in particular DNA and RNA methylation. More particularly, the present invention relates to a method of either qualitatively or quantitatively assessing, with improved sensitivity, the cytosine methylation of partially methylated DNA or RNA. The method of the present invention is useful in a range of applications including, but not limited to, the diagnosis of conditions or monitoring of developmental phenotypes which are characterised by DNA or RNA methylation changes.