Epigenetics & Chromatin

The usage of PCR-based or NGS-based liquid biopsy assays for circulating tumor DNA (ctDNA) detection is growing rapidly in clinical labs. However, problems oft en occur such as disagreements in ctDNA detection results among assays from diff erent developers. One lacking area of research is on the understanding of how plasma factors aff ect the cell-free DNA (cfDNA) extraction, a process that contributes a significant portion of total assay variation. One reason for the lack of study might be due to the fact that plasma obtained from a normal donor is often contaminated with interfering endogenous DNA. To address this issue, Anchor Molecular Inc. has developed a proprietary technology that specifi cally removes the DNA from plasma without aff ecting other composition of the plasma. Th e DNA content in the treated plasma was measured to be less than 0.01 nano grams per milliliter. All other components such as proteins and lipids remain the same as in normal patient plasma. The spiked DNA extracted from the plasma can be linearly recovered, mimicking the spiked DNA in normal plasma. Both synthetic DNA and fragmented genomic DNA showed at least 4 months of real-time stability at 2-8ËšC. Specifi city of cfDNA extraction was studied by spiking both low- and high-molecular-weight DNA fragments followed by extraction and analysis using Bioanalyzer. Th e sensitivity of cfDNA extraction was examined by extractions of the spiked low copy number of target DNA fragments. Th e result showed that diff erent cfDNA extraction kits exhibited diff erent preference in extracting DNA of diff erent sizes. Diff erence in both the quantity and the precision of the recovery of low level of cfDNA can be quantitatively compared among diff erent cfDNA extraction methods. Th e data demonstrated that quality controls samples based on DNA-free plasma can be used for ctDNA assay validation, extraction monitoring and the end-to-end process quality control for genetic analysis.