A Novel Cell-Free DNA Extraction System for Cancer Diagnostics
Poster Oct 14, 2016
Amanda Fan, Alex Yeo, Yin Kum Ng, Elian Rakhmanaliev, Pramila Ariyaratne, Charlie Lee, Gerd Michel, Tatiana Ivanova
Circulating cfDNA in human plasma becoming an important source for tumour mutation detection in cancer diagnostics. In this study we compared a column-based method and a novel automated magnetic beads-based kit (Sentosa SX cfDNA Kit (4x8)). This kit was optimized for use on a robotic liquid handling platform (Sentosa SX101). Quality of cfDNA extracted by both methods was assessed using ALU repeats qPCR assay and NGS-based Sentosa SQ CRC test. DNA was extracted from plasma samples with spiked-in fragmented (~170bp) HCT116 gDNA (KRAS G13D positive) using both methods. The ALU247/115 ratio for DNA extracted by the Sentosa kit was 0.19-0.28 and for the column-based method >0.7 (expected ratio for cfDNA <0.5 and for gDNA ~1.0). Amount and quality of DNA extracted by both methods was sufficient to prepare NGS libraries for all samples. KRAS G13D was detected in all samples extracted by the Sentosa kit but not in column-based extracted samples. The Sentosa cfDNA kit selectively extracts cfDNA over high molecular weight gDNA and appears as an efficient solution for cfDNA extraction from plasma. Integration into the qPCR- and NGS-based workflows makes the Sentosa cfDNA kit a universal diagnostics tool, which can be used in combination with various IVD assays.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE