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Ultra-Sensitive Quantification of Genome Editing Events by Droplet Digital™ PCR (ddPCR™)



February 22, 2017

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  • "For more info, visit Genome editing tools, including TALENs and the CRISPR/Cas9 system, have revolutionized our ability to edit genomes of any cell, including human induced pluripotent stem cells (iPSCs). Sequence-specific nucleases are used to induce double-strand breaks or nicks at target sites, activating the DNA repair pathways of nonhomologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ produces small insertions or deletions and is useful for disrupting gene function. HDR can induce precise gene repair of one to hundreds of base pairs, allowing for correction of point mutations and introduction of exogenous sequences. While powerful, the efficiency of gene editing is cell type–dependent and often low ( less than 5%), particularly in primary cells or iPSCs. Here we introduce Droplet Digital PCR Assay strategies sensitive enough to detect edited alleles present at a frequency less than 0.5%. These methods are useful for ultra-sensitive detection of edited alleles and offer a rapid, low-cost readout for technical optimization of genome editing protocols. Droplet Digital PCR technology is the most precise and sensitive digital PCR solution for a wide variety of applications, featuring flexible assay chemistry optimized for both hydrolysis probe and dye-based assays. Nanoliter droplet partitioning reduces bias from amplification efficiency differences and PCR inhibitors and enables exquisitely accurate target quantification in a convenient assay design, without the use of standard curves. The QX200™ Droplet Digital PCR System, Bio-Rad's second-generation digital PCR system, provides absolute quantification of target DNA or RNA molecules using EvaGreen or TaqMan hydrolysis probes, yielding unmatched sensitivity and precision for a wide variety of applications. Presenter: Jennifer R. Berman, PhD, Staff Scientist, Bio-Rad Digital Biology Center Jennifer Berman earned her PhD in Genetics at UCSF, studying the genetic basis of aging and lifespan as a Howard Hughes Medical Institute Predoctoral Fellow. Following postdoctoral work at Stanford, Dr. Berman focused on the contribution of human genetic variation to health and disease in the personal genomics space. At the Digital Biology Center, Dr. Berman leads advanced ddPCR applications and assay development projects in cancer and applied genomics."

    Analytical Techniques

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