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.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE