Person:

Mamon, Harvey

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal 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 that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four 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. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.

Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.