Abstract

DiSSeCT Technology Provides Mutation Enrichment Prior to PCR or COLD-PCR and Enables Detection of Traces of Rare Mutations in Cancer Samples

Mike Makrigiorgos, Professor, Dana-Farber Cancer Institute/Harvard Medical School

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, pre-natal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DiSSeCT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic-beads. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature, selectively separated from wild-type DNA and followed by PCR/COLD-PCR. We demonstrate multiplexed enrichment by 100-400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using 2-4 successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or High-Resolution-Melting (HRM). The simplicity, scalability and reliability of DISSECT makes it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.