title: Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing.
abstract: This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them.
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158;
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Miyaoka
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158;
authors.forename: Amanda H
authors.initials: AH
authors.lastname: Chan
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158; Departments of Medicine, and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Conklin
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D000072669:Gene Editing; D009711:Nucleotides / Q000032:analysis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D009711:Nucleotides; D004247:DNA