Extended Data Fig. 5
From: Efficient targeted insertion of large DNA fragments without DNA donors
Illustration of the strategies of real-time qPCR and In-Out PCR to quantify insertion efficiency. a, The edited samples mainly include two types of sequences. Two pairs of qPCR primers were designed to amplify the WT strands or edited strands at the LSP1 locus, respectively. The sequence between paired pegRNAs was showed in purple, and the insertion fragment was show in red. b, The WT and edited sequences were designed and synthesized. These two fragments were serially diluted from 10-2 to 10-8 ng/μL to serve as templates for standard curve. c, Two standard curves were generated by real-time qPCR using primers and templates described above. The standard curve reflects the correlation between DNA copy numbers and quantification cycle (Cq). d, Diagram of In-Out PCR for detecting inserted fragment. Two pairs of primers (P1/P2: green arrows; P3/P4 red arrows) were designed to amplify the WT and edited genomes. For the WT genome, the P1,P2 and P4 but not P3 could bind to the genome sequence. For the edited genome, the two pairs of primers can amplify both WT and edited sequences. e, Detection of the precise insertion of 150 bp fragments at five endogenous loci by In-Out PCR. The expected products of P1/P2 and P3/P4 were pointed by green arrows and red arrows, respectively (n = 2 independent experiments).
