Compartmentalized ecDNA Detection via Sequence-Specific Cleavage
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Solution Overview
Problem
Existing methods for detecting extrachromosomal DNA (ecDNA) are complex, time-consuming, and inefficient, making it difficult to distinguish between chromosomal and ecDNA, particularly in the context of cancer research where ecDNA is associated with poor prognosis.
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 detection of compartments containing both sequences, allowing for efficient identification of ecDNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next generation sequencing or FISH is used to detect ecDNA, then detection capability is achieved, but the process becomes complex and time-consuming
Solution Approach 1:
The patent extracts the ecDNA detection function from complex whole-genome sequencing processes by designing specific primer sets that target only ecDNA-related sequences. This allows selective amplification and detection of ecDNA without the need for comprehensive sequencing, thereby simplifying the detection process while maintaining detection capability
Solution Approach 2:
The detection method is divided into distinct segments: (a) specific cleavage of the third sequence in the target nucleotide sequence, (b) separation into compartments, and (c) fluorescent detection. This segmentation allows each step to be optimized independently and performed in a standardized workflow, reducing overall process complexity
2Measurement precision
If conventional ecDNA detection methods are used, then detection is possible, but it is difficult to distinguish between chromosomal and ecDNA
Solution Approach 1:
The patent applies local quality by designing the target nucleotide sequence with specific regional characteristics: a first sequence, a third sequence, and a second sequence arranged in a particular configuration. The third sequence serves as a unique marker that is present in ecDNA but not in chromosomal DNA, allowing local differentiation and accurate identification of ecDNA molecules
Solution Approach 2:
The patent introduces an intermediary cleavage step using restriction enzymes or other nucleases that specifically cut the third sequence. This intermediary action converts the presence of the third sequence into a detectable physical change (cleavage products), which serves as a mediator between the ecDNA structure and the final detection signal, enabling clear distinction from chromosomal DNA
3Reliability
If comprehensive ecDNA analysis is performed, then complete characterization is achieved, but cost and time requirements increase
Solution Approach 1:
The patent performs preliminary action by pre-designing and pre-validating specific primer sets and probe combinations that target conserved ecDNA sequences. This preliminary preparation allows the actual detection process to proceed directly to amplification and detection without requiring extensive optimization or validation during sample analysis, thereby reducing detection time while maintaining reliability
Solution Approach 2:
The method employs parameter changes by optimizing PCR conditions, probe concentrations, and cleavage reaction parameters to achieve rapid amplification and detection. By adjusting these parameters, the method achieves reliable ecDNA detection in a shortened time frame compared to conventional sequencing-based approaches
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 and cost-effective detection of ecDNA by distinguishing it from chromosomal DNA, facilitating early cancer detection and monitoring.
Implementation Method 1
a treatment of sequence-specifically cleaving a third sequence present between a first sequence and a second sequence in a target nucleotide sequence
Implementation Method 2
detecting a compartment containing DNA having both the first sequence and the second sequence in the plurality of compartments after the step (b)
Data Source
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) 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) separating the sample after (a) into a plurality of compartments; and (c) detecting a compartment containing DNA having both the first sequence and the second sequence in the plurality of compartments after (b).