qPCR DNA Methylation Detection Kit
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Solution Overview
Problem
Current DNA methylation detection methods, such as bisulfite sodium treatment and methylation-sensitive restriction endonuclease treatment, face challenges including inaccurate results due to artificial heterogeneity, DNA breakage, complex procedures, high costs, and limited specificity, making them inefficient and unsuitable for early cancer detection.
Innovation Solution
A method and kit based on quantitative polymerase chain reaction (qPCR) that uses a Taq enzyme premix system, primers, and probes to detect DNA methylation by differentiating between methylated and non-methylated DNA through specific PCR conditions, allowing for accurate and sensitive detection of DNA methylation levels without the need for bisulfite treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sodium treatment is used for DNA methylation detection, then methylation detection can be performed, but artificial heterogeneity is introduced resulting in inaccurate results
Solution Approach 1:
The patent extracts and eliminates the bisulfite treatment step from the detection process. By using a methylation-sensitive restriction endonuclease that directly recognizes and cuts methylated DNA sequences, the method removes the source of artificial heterogeneity while maintaining detection capability, thus improving result accuracy without the need for bisulfite conversion.
Solution Approach 2:
The patent introduces a methylation-sensitive restriction endonuclease as an intermediary enzyme that specifically interacts with methylated DNA. This enzyme serves as a mediator that directly detects methylation status through its selective cutting activity, avoiding the need for bisulfite treatment and subsequent heterogeneous artifacts.
2Measurement precision
If bisulfite sodium treatment is used for DNA methylation detection, then methylation detection can be performed, but DNA breakage occurs affecting determination
Solution Approach 1:
The patent removes the bisulfite treatment step that causes DNA breakage. By using a restriction endonuclease-based approach that operates under milder conditions and does not require chemical conversion, the method preserves DNA integrity while maintaining detection precision.
Solution Approach 2:
The patent converts the detection problem into a benefit by using the natural specificity of restriction endonucleases for methylated sequences. Instead of using harsh chemical treatment that damages DNA, the method leverages the enzyme's natural recognition and cutting specificity to achieve both DNA integrity preservation and accurate methylation detection.
3Measurement precision
If bisulfite sodium treatment and downstream platforms are used for DNA methylation detection, then detection can be performed, but procedures become complicated and time-consuming
Solution Approach 1:
The patent merges the methylation detection function directly into the PCR amplification process itself. By designing primers and probes that specifically recognize methylated sequences, the method combines methylation status determination with DNA amplification, eliminating the need for separate bisulfite treatment and downstream analysis steps, thus simplifying the overall procedure.
Solution Approach 2:
The patent creates a universal detection system where a single qPCR-based assay simultaneously performs DNA amplification and methylation status determination. The primers and probes are designed to work directly with methylated DNA, making the system multi-functional and eliminating the need for multiple separate processes.
4Measurement precision
If methylation-sensitive restriction endonuclease treatment is used for DNA methylation detection, then specific site detection is achieved, but regions without specific sites cannot be detected
Solution Approach 1:
The patent designs primers and probes that can bind to methylated sequences throughout the entire gene region, not just at specific restriction sites. This universal binding capability allows detection of methylation status across any region of the target gene, making the system versatile for detecting methylation in regions with or without specific restriction enzyme recognition sites.
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
The qPCR-based method provides simple, rapid, and reliable detection of DNA methylation, suitable for various methylation levels, with high sensitivity and specificity, overcoming the limitations of existing methods by directly measuring methylation levels without causing DNA breakage or requiring complex procedures.
Implementation Method 1
quantitative polymerase chain reaction (qPCR)... a Taq enzyme premix system (Taq enzyme, buffer, and dNTP)
Implementation Method 2
high-temperature denaturation amplification and low-temperature denaturation amplification
Data Source
AI summary
Provided is a method and kit for detecting DNA methylation based on quantitative polymerase chain reaction (qPCR). The present disclosure achieves the detection of DNA methylation based on qPCR, where PCR is used to prepare fragments of different DNA methylation levels through base incorporation, and then the fragments are subjected to qPCR detection with two programs respectively to get ΔCt and establish a correspondence between ΔCt and a DNA methylation level. The method evaluates a DNA methylation level of a target gene by detecting ΔCt of the target gene. The present disclosure establishes a new convenient method for detecting a DNA methylation difference. According to verification results of experiments with lung cancer and colorectal cancer plasma samples, the method has simple operations, short detection time, and accurate and reliable results, is suitable for the detection of all types of methylated DNA fragments, and has high application values.