Cell Design Studio

Digital PCR Applications in Sigma-Aldrich Custom Cell Line Engineering

Sigma-Aldrich Cell Design Studio leverages CompoZr® Zinc Finger Nuclease technology to create precise genetic modifications in mammalian cell lines. Zinc finger nucleases (ZFNs) consist of a DNA-binding domain that recognizes a unique sequence of DNA and a DNA-cleaving domain that generates double-strand breaks at the recognized sequence. Double-strand breaks may be repaired by natural DNA repair processes including Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Possible genetic modifications include targeted gene disruption resulting in complete gene knockouts or targeted gene insertion resulting in knock-in of gene mutations, promoters, or reporters. The implementation of BioRad QX100™ Droplet Digital™ PCR (ddPCR) instrumentation complements the custom cell engineering workflow. Droplet Digital PCR is used to characterize the expected frequency of homologous recombination and develop a screening strategy based on this expected frequency. In some cell lines homologous recombination occurs at a low frequency. In such a case we have utilized ddPCR to screen cell pools totaling approximately 175,000 cells in order to identify four unique clones having the desired mutation. Digital PCR is also used to accurately and expediently measure target gene copy number, including in polyploid cell lines. In the final step of cell line engineering ddPCR has proven useful to ensure clonality of cell lines. The introduction of ddPCR into the cell line engineering workflow has improved and accelerated custom cell line completion.

Figure 1. Detection and determination of relative copy number of SNP mutation with droplet digital PCR.

A mutation-specific probe was prepared carrying the fluorescent dye FAM, and the probe for unmodified (wild type) sequence carried the fluorophore HEX. The X axis is the HEX signal, generated as a function of the presence of wild type sequence. The Y axis is the FAM signal, generated as a function of the presence of the SNP mutation sequence. Both modified and wild type sequences were detected in the target DNA and the relative abundance of each sequence could be directly determined.

Figure 2. Pooled screening strategy for rare mutational event detection using digital droplet PCR.

Figure 3. Isolation of clones harboring rare mutational event using digital droplet PCR.