Methylation-Sensitive Endonuclease DNA Methylation Detection
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Solution Overview
Problem
Current methods for DNA methylation analysis, such as bisulfite sequencing and Methylation Specific PCR, are labor-intensive, expensive, and prone to contamination due to their complexity and multi-step processes.
Innovation Solution
A method involving a reaction mixture with a methylation-sensitive endonuclease (MSE) and DNA polymerase, where DNA samples are incubated under conditions for cleavage and PCR, allowing for single-step selective amplification and quantification of methylated DNA, reducing the risk of contamination and increasing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used for DNA methylation analysis, then methylation status can be detected, but the process becomes labor intensive and complicated
Solution Approach 1:
The patent combines multiple steps (DNA denaturation, MSE digestion, and PCR amplification) into a single reaction mixture and single incubation step. The MSE enzyme, DNA polymerase, primers, and buffer are all present together in one reaction tube, eliminating the need for separate bisulfite treatment and multiple processing steps while maintaining methylation detection accuracy.
Solution Approach 2:
The single reaction mixture serves multiple functions: it provides the MSE enzyme for methylation-sensitive digestion, the DNA polymerase for amplification, the primers for specific target amplification, and the buffer for maintaining optimal conditions. This multi-functional system replaces the specialized separate reagents and steps of traditional methods.
2Measurement precision
If traditional multi-step methods are used for DNA methylation analysis, then methylation can be detected, but the risk of contamination increases
Solution Approach 1:
By performing digestion and amplification in a single closed reaction tube without opening or transferring samples between steps, the patent eliminates contamination risks associated with multiple handling steps. The reaction mixture remains sealed throughout the entire process, maintaining reliability while detecting methylation status.
3Measurement precision
If current DNA methylation analysis methods are used, then methylation status can be determined, but time consumption increases
Solution Approach 1:
The patent enables continuous action by performing digestion and amplification simultaneously in the same reaction mixture without interruption or intermediate steps. The MSE enzyme digests unmethylated DNA while the polymerase amplifies methylated DNA templates in an uninterrupted continuous process, significantly reducing analysis time while maintaining accuracy.
4Measurement precision
If conventional methylation analysis methods are used, then DNA methylation can be analyzed, but cost increases due to expensive machinery
Solution Approach 1:
The patent uses a simple, disposable reaction mixture that can be prepared in standard PCR tubes without requiring expensive specialized machinery like HPLC or mass spectrometers. The entire analysis is performed using conventional PCR equipment already available in most laboratories, dramatically reducing cost while maintaining analytical capability.
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 enables rapid, accurate, and cost-effective quantification of DNA methylation, integrating multiple steps into a single reaction, thereby improving the reliability and speed of methylation analysis.
Implementation Method 1
contacting a DNA sample with a reaction mixture comprising a methylation-sensitive endonuclease (MSE)... incubating the sample under conditions permissive for DNA cleavage by the MSE
Implementation Method 2
incubating the sample under conditions permissive for DNA polymerization (i.e., conditions permissive for polymerase chain reaction (PCR))
Data Source
AI summary
In a first aspect, the invention concerns a method for detecting or quantifying DNA methylation at a locus. In one embodiment, a methylation-sensitive endonuclease is formulated together with a polymerase enzyme in an appropriate reaction mixture such that amplification of DNA occurs in a methylation specific manor. Quantitative DNA amplification at selected loci can be used to determine the level of methylation. Kits and reagents for performing such methods are also provided.