Improvement of digital PCR conditions for direct detection of KRAS mutations

Abstract

Background In standard analytical conditions, an isolation step is essential for circulating tumor DNA (ctDNA) analysis. The necessity of this step becomes unclear with the development of highly sensitive detection methods. The aim of this study was to evaluate ctDNA mimetic nDNA detection as reference materials (RMs) using dPCR technologies either directly from serum or without serum. Methods To determine an absolute count of both mutation and wild‐type bearing DNA molecules, genomic DNA (gDNA) and nucleosomal DNA (nDNA), which are similar in size to cell‐free DNA, were evaluated. We tested 3 KRAS mutations in colorectal cancer cell lines. Results We describe the recent progress in RMs. The short DNA fragments, such as sDNA and nDNA, exhibited higher quantitative values of dPCR compared to gDNA. The efficiency between Atlantis dsDNase (AD) and Micrococcal Nuclease (MN) affects DNA quantification. Moreover, there was a significant difference in dPCR output when spiking gDNA or nDNA containing KRAS mutations into FBS compared to the dPCR output under non‐FBS conditions. Conclusion The matrix effect crucially affects the accuracy of gDNA and nDNA level estimation in the direct detection of mimic of patient samples. The form of reference material we proposed should be optimized for various conditions to develop reference materials that can accurately measure copy number and verify the detection of KRAS mutations in the matrix.

TP53 Mutation in Circulating exoDNA Correlates with Outcomes of Patients with Hepatocellular Carcinoma.

“…Unfortunately, the diagnostic e cacy of traditional markers, such as AFP, is not ideal [3]. In recent years, multiple studies have used circulating free DNA (cfDNA) from the blood of pancreatic and colorectal cancer patients to identify tumor-speci c mutations as cancer markers [4,5]. However, cfDNA originates from dead cells in damaged tissue or accumulates as a result of physiological cell turnover [6,7].…”