Circular DNA Analysis via Enzymatic Linearization
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Solution Overview
Problem
Current methods for analyzing circular DNA in biological samples, such as plasma, are limited in their ability to accurately differentiate and quantify circular and linear forms of mitochondrial DNA, which hinders the detection of diseases like cancer, as they often misinterpret degraded linear mtDNA as circular mtDNA.
Innovation Solution
The use of restriction enzymes and transposases to linearize circular DNA, followed by sequencing and bioinformatic analysis to identify and characterize circular DNA molecules by mapping reversed end sequences and detecting cutting tags, allows for the differentiation and analysis of circular and linear forms of mtDNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to analyze circular DNA in plasma, then analysis can be performed, but circular and linear forms of mitochondrial DNA cannot be accurately differentiated and quantified
Solution Approach 1:
The patent divides the analysis into two distinct pathways: one for circular DNA molecules (which remain intact) and one for linear DNA molecules (which are fragmented). By treating these forms separately through size-based separation and form-specific enzymatic treatments, the method achieves accurate differentiation and quantification of circular versus linear mtDNA, directly resolving the measurement precision issue.
Solution Approach 2:
The patent applies preliminary enzymatic treatments (exonucleases, restriction enzymes, or transposases) to selectively modify linear DNA molecules before analysis. These preliminary actions convert linear DNA into a distinguishable form or remove it selectively, enabling subsequent accurate identification and quantification of circular DNA forms that would otherwise be indistinguishable from degraded linear mtDNA.
2Reliability
If degradation of linear mtDNA is misinterpreted as circular mtDNA, then false positive results occur, but this misinterpretation cannot be prevented by conventional analysis methods
Solution Approach 1:
The patent introduces size-based separation techniques (such as gel electrophoresis or size-exclusion chromatography) as an intermediary step between DNA extraction and analysis. This intermediary process physically separates circular DNA molecules from linear DNA fragments based on their different migration patterns or elution characteristics, preventing misinterpretation and enabling accurate identification of genuine circular DNA forms.
Solution Approach 2:
The patent changes the physical and chemical parameters of DNA analysis by using form-specific enzymatic treatments that differentially affect circular versus linear DNA. By adjusting enzyme type, concentration, and incubation conditions, the method creates distinct analytical signals for each DNA form, thereby improving both identification accuracy and reliability of disease detection.
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 precise identification and quantification of circular DNA forms, improving the detection of diseases by providing accurate genetic and methylation data, enhancing diagnostic capabilities.
Implementation Method 1
The use of restriction enzymes and transposases to linearize circular DNA
Data Source
AI summary
Techniques are provided for analyzing circular DNA in a biological sample (e.g., including cell-free DNA, such as plasma). For example, to measure circular DNA, cleaving can be performed to linearize the circular DNA so that they may be sequenced. Example cleaving techniques include restriction enzymes and transposases. Then, one or more criteria can be used to identify linearized DNA molecules, e.g., so as to differentiate from linear DNA molecules. An example criterion is mapping a pair of reversed end sequences to a reference genome. Another example criterion is identification of a cutting tag, e.g., associated with a restriction enzyme or an adapter sequence added by a transposase. Once circular DNA molecules (e.g., eccDNA and circular mitochondrial DNA) are identified, they may be analyzed (e.g., to determine a count, size profile, and/or methylation) to measure a property of the biological sample, including genetic properties and level of a disease.