Publication: Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment
Open/View Files
Date
2016
Published Version
Journal Title
Journal ISSN
Volume Title
Publisher
Oxford University Press
The Harvard community has made this article openly available. Please share how this access benefits you.
Citation
Song, Chen, Yibin Liu, Rachel Fontana, Alexander Makrigiorgos, Harvey Mamon, Matthew H. Kulke, and G. Mike Makrigiorgos. 2016. “Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment.” Nucleic Acids Research 44 (19): e146. doi:10.1093/nar/gkw650. http://dx.doi.org/10.1093/nar/gkw650.
Research Data
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.
Description
Other Available Sources
Keywords
Terms of Use
This article is made available under the terms and conditions applicable to Other Posted Material (LAA), as set forth at Terms of Service