Figure 4
From: Rapid, precise quantification of large DNA excisions and inversions by ddPCR
Design and validation of precise excision assay. (a) Schematic of precise excision assay. A probe is designed to align to the predicted excision junction. The probe can be duplexed with the general ddXR probe or a reference probe for normalization (not shown). (b) Percent of alleles with total and precise excision in population and clonal lines, presented as mean of triplicate assays ± S.D. (c) ddXR quantification in samples with defined frequency of precise excision. Duplicate samples were prepared by serial dilution of precise excision clonal DNA with imprecise excision clonal DNA. Presented as percent of excision alleles with precise repair versus expected, R2 = 0.98. (d, e) 2D plots of genomic DNA spiked with 1:1 mixtures of HPRT1 excision reporter plasmids with precise and imprecise excision. Alleles with precise excision are positive for FAM and HEX (blue) while alleles with either type of imprecise excision are positive for HEX only (green). Measured ratio of precise to 1 bp mismatch was 0.952 (d, Poisson range 0.996–0.909) and of precise to 1 bp deletion was 0.919 (e, Poisson range 0.961–0.878).
