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

Cold Spring Harb Protoc. 2016 Aug 1;2016(8). doi: 10.1101/pdb.prot086801.

Authors

Yuichiro Miyaoka¹, Amanda H Chan¹, Bruce R Conklin²

Affiliations

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

PMID: 27250210
DOI: 10.1101/pdb.prot086801

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.

MeSH terms

DNA / chemistry*
DNA / genetics*
Gene Editing*
Nucleotides / analysis*
Nucleotides / genetics*
Polymerase Chain Reaction / methods*

Substances

Nucleotides
DNA