Apoptosis-Induced ctDNA Reference Samples for Assay Calibration
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Solution Overview
Problem
The development of well-characterized reference samples for circulating tumor DNA (ctDNA) is complicated by the degraded nature of DNA in blood, making it difficult to validate the performance of detection methods for somatic mutations, as clinical samples are limited and difficult to obtain.
Innovation Solution
A method involving the induction of apoptosis in tumor cells using apoptosis inducers like camptothecin (CPT) to extract ctDNA, which simulates the fragmentation pattern of plasma ctDNA and can be used as quality control materials without affecting the detection of point mutations, copy number variations, and structural variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If clinical samples are used as reference samples for ctDNA detection validation, then the samples reflect true clinical performance, but the samples are limited in availability and difficult to obtain long-term
Solution Approach 1:
The patent creates artificial reference samples by inducing apoptosis in tumor cells to generate ctDNA that copies the fragmentation patterns and characteristics of clinical ctDNA. This allows unlimited production of reference samples that maintain the authentic properties needed for detection method validation without relying on limited clinical samples
2Ease of manufacture
If synthetic ctDNA reference samples are developed to replace clinical samples, then the samples become simple to prepare and available in unlimited quantities, but the samples must maintain high consistency with clinical samples
Solution Approach 1:
The patent induces apoptosis in tumor cells using controlled parameters (apoptosis inducers, incubation time, temperature) to generate ctDNA with specific fragmentation patterns that match clinical samples. By optimizing these parameters, the method achieves both ease of preparation and high consistency in fragment size distribution (150-200 bp range) with authentic clinical ctDNA
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
The method provides a reliable and consistent source of ctDNA reference samples that mimic real plasma ctDNA fragmentation patterns, enabling accurate validation of detection methods and quality control in assays, with potential applications in cancer diagnosis and monitoring.
Implementation Method 1
inducing apoptosis in tumor cells by treating tumor cells with CPT at a concentration of about 10 μM for 2-8 hours (e.g., about 5 hours)
Data Source
AI summary
The present disclosure relates to methods of preparing circulating tumor DNA (ctDNA) reference samples including: inducing apoptosis in tumor cells to obtain DNA fragments and then extracting DNA from the tumor cells to obtain the circulating tumor DNA reference samples. The methods to prepare ctDNA reference samples disclosed herein are simple and easy to use, suitable for various tumor cells, and the variant information can be retained to simulate the ctDNA in animals. In some embodiments, the reference samples can facilitate assay calibration and evaluation.