Methylation-Sensitive Restriction Endonuclease DNA Quantification
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Solution Overview
Problem
Current methods for analyzing DNA methylation patterns in cancer cells, particularly in colorectal cancer, face challenges such as the need for high starting DNA concentrations and the occurrence of false positives due to incomplete digestion by methylation-sensitive restriction endonucleases, which require relative quantification against an undigested internal control sample.
Innovation Solution
A method involving the use of two or more methylation-sensitive restriction endonucleases for complete digestion of DNA at 4-6 pgDNA/unit of endonucleases per hour, followed by quantitative PCR amplification using primers flanking the methylation-specific restriction endonuclease recognition sequences, allowing for absolute quantification of methylated DNA regions without the need for an undigested internal control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If methylation-sensitive restriction endonucleases are used for DNA digestion, then DNA methylation analysis can be performed, but incomplete digestion occurs leading to false positives and requiring relative quantification against undigested internal control
Solution Approach 1:
The patent optimizes the digestion parameters by using two or more methylation-sensitive restriction endonucleases in combination, increasing the enzyme units per hour to achieve complete digestion. This parameter change eliminates incomplete digestion and the need for internal control samples, resolving the contradiction between reliability and device complexity.
Solution Approach 2:
The patent divides the digestion process into multiple enzyme treatments instead of using a single endonuclease. By segmenting the digestion function across multiple specialized enzymes, complete digestion is achieved more reliably, eliminating false positives and the need for complex internal control systems.
2Measurement precision
If high starting DNA concentrations are required for methylation analysis, then accurate quantification can be achieved, but the method becomes less sensitive for low DNA input samples
Solution Approach 1:
The patent changes the digestion parameters by using multiple endonucleases with higher combined units per hour, which improves digestion efficiency and allows accurate quantification at lower starting DNA concentrations. This resolves the contradiction between measurement precision and adaptability to low input samples.
3Reliability
If relative quantification against undigested internal control is used, then methylation levels can be assessed, but the method becomes more complex and less cost-effective
Solution Approach 1:
The patent extracts and eliminates the undigested internal control sample from the methodology by achieving complete digestion through optimized enzyme conditions. This removes the unnecessary complexity and cost associated with preparing and analyzing internal controls while maintaining reliable methylation assessment.
Solution Approach 2:
By changing the enzyme units per hour parameter to achieve complete digestion, the patent eliminates the need for internal control samples, thereby simplifying the workflow and reducing costs while maintaining reliable methylation level assessment.
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 simplifies and enhances the accuracy of DNA methylation analysis by achieving complete digestion and eliminating the requirement for relative quantification, providing a more reliable and cost-effective method for screening DNA methylation levels in biological samples.
Implementation Method 1
contacting DNA from said biological sample with two or more methylation-sensitive restriction endonucleases and digesting said DNA
Implementation Method 2
quantitatively amplifying the digested DNA sample of step (i) using one or more forward primers and one or more reverse primers
Data Source
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AI summary
The present invention relates to a method of screening for the presence of methylated DNA in a biological sample by using multiple methylation-sensitive restriction endonucleases. More particularly, the present invention relates to a method of quantitatively screening for the level of one or more methylated genes of interest without the requirement that an undigested internal reference sample is used as a point of reference against which relative quantification is calculated. The present invention is useful in a range of applications including, but not limited to, providing a simpler and more accurate means to determine DNA methylation status, such as in the context of diagnosing or monitoring conditions characterised by changes to DNA methylation.