Exogenous DNA Calibrator for Circulating DNA Quantification
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Solution Overview
Problem
Current methods for extracting and quantifying circulating DNA lack standardization, leading to variability in results and making it difficult to establish reliable diagnostic and prognostic tools, particularly for cancer detection and monitoring of physiological states.
Innovation Solution
A method involving the addition of exogenous DNA fragments as calibrators during extraction, followed by digital PCR or NGS analysis, to standardize the extraction and quantification of circulating DNA, ensuring reproducibility and accurate measurement of DNA concentrations and size profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If circulating DNA extraction methods are used without standardization, then the extraction process is simple and accessible, but the measurement precision and reliability of results deteriorate due to variability in DNA concentration and size profile
Solution Approach 1:
The invention changes the parameter of DNA fragment size to a standardized range (50-200 base pairs) for the exogenous calibrator, which matches the size profile of circulating DNA. This parameter matching enables accurate quantification while maintaining extraction simplicity, resolving the contradiction between ease of extraction and measurement precision.
Solution Approach 2:
The invention introduces an exogenous DNA fragment as an intermediary calibrator that is added to the sample before extraction. This intermediary serves as a reference standard to quantify the extraction efficiency and normalize results across different samples and experiments, thereby improving measurement precision without complicating the extraction procedure.
2Measurement precision
If exogenous DNA calibrators are added to standardize extraction, then measurement precision and reliability improve, but the complexity of the extraction procedure increases
Solution Approach 1:
The exogenous DNA calibrator is added to the sample before the extraction process begins (preliminary action). This allows the calibrator to undergo the same extraction steps as the circulating DNA, enabling accurate normalization of extraction efficiency without requiring separate calibration procedures or additional equipment.
Solution Approach 2:
The invention merges the quantification of circulating DNA and exogenous calibrator into a single extraction and analysis process. Both DNA types are extracted simultaneously using the same protocol and quantified together, simplifying the overall procedure while maintaining high measurement precision through the calibrator reference.
3Ease of manufacture
If the size profile of circulating DNA is not maintained during extraction, then the extraction process is simpler, but the reliability of biological validation deteriorates due to loss of small fragments
Solution Approach 1:
The invention standardizes the size parameter of the exogenous calibrator to 50-200 base pairs, which matches the typical size of circulating DNA fragments. This parameter alignment ensures that the calibrator experiences the same size-dependent extraction efficiency as the circulating DNA, allowing accurate normalization and maintaining the size profile reliability without complicating the extraction.
Data Source
Figure 1a~1b
Figure 2a
Figure 2b
AI summary
The invention relates in particular to a method for detecting and/or quantifying cell-free DNA from a sample of biological fluid of a patient of interest, comprising at least: (i) a step of extracting cell-free DNA from a sample of biological fluid to which at least one effective quantity of a first exogenous DNA fragment having 50-2000 base pairs, preferably 50-200 base pairs, preferably 60-160 base pairs, even more preferably 70-150 base pairs and better still 80-140 base pairs (ICE), is added; (ii) a step of amplifying and quantifying the cell-free DNA extracted in step (i) and the exogenous DNA fragment ICE; and (iii) a step of standardising the amount of cell-free DNA extracted, comprising the calculation of a first ratio (Grewis) of the number of copies of cell-free DNA to the number of copies of the first fragment of exogenous DNA (ICE), and the uses thereof for the purpose of diagnosis, prognosis or theragnosis, or for monitoring the progress of a specific physiological state of a patient of interest likely to release circulating DNA.