Dr David Boucher

david_promega

Applications Specialist/Territory Manager, Promega Corporation

Presentation Title: Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection

A major roadblock to using circulating cell free DNA (ccfDNA) for cancer biomarker detection is the diffıculty of obtaining a pure sample. Contaminating genomic DNA (gDNA) can overwhelm already rare events within a CCF sample and prevent sensitive detection of these key mutations. Unfortunately, liquid biological samples often are not collected and processed in an ideal manner, resulting in decreased quality and purity. Many studies report diffıculties in detecting mutations in ccfDNA which may originate from variable levels of gDNA background based how samples are processed. To this end, we present an effective method for purifying relevant small nucleic acids from larger genomic species after DNA purifıcation from plasma. Using a simple magnetic bead based technique, we demonstrate effıcient removal of gDNA even in highly contaminated samples. This method can be used to enrich liquid biological samples for improved detection of rare genetic events such as microsatellite instability and mutations in key oncogenes. Using plasma from three individuals with colorectal cancer tumors harboring known mismatch repair gene mutations, we compare detection of somatic mutations in ccfDNA before and after this gDNA clean up method. Microsatellite instability profıles were obtained from ccfDNA and compared to normal and tumor profıles to determine if evidence of mismatch repair abnormalities could be detected. Targeted amplicon sequencing was then performed to detect tumor associated mutations. DNA isolated from matched adjacent normal and tumor tissues were used as controls in these experiments to confırm mutations were of tumor not germline mutations. Effectively removing gDNA contamination from ccfDNA samples can improve biomarker detection consistency from liquid biological samples. We demonstrate a magnetic bead-based method for ccfDNA clean-up, which can be used prior to microsatellite instability detection by fragment analysis or rare variant detection by next generation sequencing.