Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing

Yuichiro Miyaoka; Amanda H. Chan; Bruce R. Conklin

doi:10.1101/pdb.prot086801

Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing

¹Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158;
²Departments of Medicine, and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143

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.