DNA Methylation and Mutation Co-Detection by Hybrid Capture
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Solution Overview
Problem
Existing methods cannot simultaneously detect methylation and mutation states in DNA samples due to the limitations of conventional sequencing technologies, which fail to distinguish methylated and non-methylated C bases, and require separate hybrid capture systems for each detection, leading to inefficiencies and high costs.
Innovation Solution
A method involving adapter ligation, enzyme digestion with methylation-sensitive restriction endonucleases, and hybrid capture using probes that target both methylation and mutation sites, allowing simultaneous detection in a single hybrid capture system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing technologies are used to detect methylation, then methylation state can be detected, but mutation state cannot be detected simultaneously due to inability to distinguish methylated and non-methylated C bases
Solution Approach 1:
The adapter ligation product sample is divided into two parts: one part undergoes enzyme digestion with methylation-sensitive restriction endonucleases for methylation detection, while the other part is preserved for mutation detection. This segmentation allows both detection types to proceed simultaneously without interference.
Solution Approach 2:
Methylation-sensitive restriction endonucleases are introduced as intermediaries to selectively digest unmethylated DNA sequences. These enzymes serve as a bridge between the DNA sample and the detection system, enabling differentiation of methylation states while preserving the original sequence information for subsequent mutation analysis.
2Measurement precision
If separate hybrid capture systems are used for methylation and mutation detection, then each detection can be performed accurately, but the experimental process becomes complex and costly
Solution Approach 1:
The patent merges methylation detection and mutation detection into a single hybrid capture system. By dividing the adapter ligation product sample into two parts and processing them differently before combining them in one hybrid capture system, the patent eliminates the need for separate systems while maintaining detection accuracy for both methylation and mutation states.
Solution Approach 2:
The hybrid capture system is designed to perform multiple functions: it can detect both methylation states (through the enzyme-digested portion) and mutation states (through the undigested portion) within the same system. This multi-functionality reduces overall system complexity and enables simultaneous detection of multiple DNA characteristics.
3Measurement precision
If separate hybrid capture systems are used for methylation and mutation detection, then each detection can be performed accurately, but detection time and reagent costs increase
Solution Approach 1:
The patent combines methylation and mutation detection workflows into a single hybrid capture system, allowing both types of detection to occur simultaneously rather than sequentially. This merging of workflows reduces total detection time and eliminates the need for separate reagent preparations and processing steps.
Solution Approach 2:
The adapter ligation product sample is divided into two parts at an early stage, and methylation-sensitive restriction endonuclease treatment is performed preliminarily on one part before hybrid capture. This preliminary differentiation allows both methylation and mutation detection to proceed in parallel during the hybrid capture step, improving overall detection efficiency.
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 reduces detection time, simplifies the experimental process, increases sample utilization, and lowers reagent costs while achieving accurate co-detection of methylation and mutation states in DNA samples.
Implementation Method 1
treating the first adapter ligation product sample with one or more methylation-sensitive restriction endonucleases, such that the DNA molecule is capable of being subjected to an enzyme digestion reaction when methylation is not present at a recognition site
Implementation Method 2
making a probe group including one or more mutation capture probes and one or more methylation capture probes in contact with the amplification product mixture to obtain a capture product
Data Source
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Figure 2A~3B
Figure 4A~4C
AI summary
Provided herein is a method for detecting methylation and mutation states of a DNA sample, and particularly, provided is a method for simultaneously detecting a methylation state and a mutation state of a DNA molecule in a sample. The method includes treating a sample with a methylation-sensitive restriction endonuclease; using a probe group including a mutation capture probe and a methylation capture probe to carry out hybrid capture on amplification products of the sample treated with the restriction endonuclease and a sample untreated with enzyme digestion; and sequencing a capture product.