title: Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Yibin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Fontana
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Makrigiorgos
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Harvey
authors.initials: H
authors.lastname: Mamon
authors.affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Kulke
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA mmakrigiorgos@partners.org.
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D008297:Male; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D057188:Workflow
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA; D004720:Endonucleases