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

Cold Spring Harb Protoc

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

Published: August 2016

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.

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http://dx.doi.org/10.1101/pdb.prot086801DOI Listing

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