ecDNA Detection by Sequence-Specific Cleavage and Compartment Screening
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Solution Overview
Problem
Current methods for detecting extrachromosomal DNA (ecDNA) are time-consuming, costly, and complex, with limitations in sensitivity and analysis complexity.
Innovation Solution
A method involving sequence-specific cleavage of a third sequence between first and second sequences in a target nucleotide sequence, followed by compartmentalization and fluorescent dye detection of compartments containing both first and second sequences, with optional nucleic acid amplification and exclusion of false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next generation sequencing or FISH methods are used to detect ecDNA, then detection capability is achieved, but the time required is extended and cost increases
Solution Approach 1:
The invention extracts and utilizes specific structural features of ecDNA (circular structure and characteristic nucleotide sequences) to design targeted detection assays. By focusing on these specific features rather than进行全面 sequencing, the method achieves rapid detection within hours while maintaining accuracy, directly resolving the time-cost contradiction
Solution Approach 2:
The invention changes the detection parameters from comprehensive genomic analysis (next generation sequencing) to targeted molecular detection (PCR-based methods detecting specific nucleotide sequences and circular structures). This parameter change enables results to be obtained in hours rather than days, while also reducing equipment and operational costs
2Measurement precision
If comprehensive ecDNA detection methods are used, then detection accuracy is improved, but analysis complexity increases
Solution Approach 1:
The invention segments the ecDNA detection process into distinct modular steps: (1) DNA extraction, (2) circular DNA enrichment, (3) PCR amplification of specific regions, and (4) analysis. Each step focuses on a specific aspect of ecDNA detection, simplifying the overall complexity while maintaining high accuracy through systematic progression
Solution Approach 2:
The invention performs preliminary enrichment of circular DNA structures before detection through specific treatment steps that isolate ecDNA from linear chromosomal DNA. This preliminary action simplifies subsequent analysis by pre-concentrating the target material and removing background interference, reducing overall analytical complexity
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
Enables rapid, cost-effective detection of ecDNA with improved sensitivity and simplicity, allowing for accurate quantification of target ecDNA.
Implementation Method 1
the step (a) may be carried out by using a guide RNA and an RNA-induced nuclease that targets the third sequence
Implementation Method 2
in the step (c), a first fluorescent dye may be used to detect a compartment having the first sequence, and a second fluorescent dye is used to detect a compartment having the second sequence
Data Source
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AI summary
According to an embodiment, there is provided a method of detecting a target ecDNA in a sample containing chromosomal DNA and ecDNA, where the method includes (a) a step of subjecting the DNA in the sample to a treatment of sequence-specifically cleaving a third sequence present between a first sequence and a second sequence in a target nucleotide sequence included in the target ecDNA, (b) a step of separating the sample after the step (a) into a plurality of compartments; and (c) a step of detecting a compartment containing DNA having both the first sequence and the second sequence in the plurality of compartments after the step (b).