Refinement of cbPCR.

<p>(A) PCR of Plpp2 locus was performed using the indicated primer sets. When F-out was absent, Plpp2 inner amplicon was detected in wild type DNA (lane 1), but was barely detected when doing cbPCR (lane 3). (B) Decreasing F-out primer length enabled detection of inner amplicons in cbPCR using wild type DNA and discrimination of mutant gDNA. (C) Using the optimized primers, completely mutant gDNA was discriminated from wild type or heterozygous mutant gDNA. Bands with expected sizes are marked with dots. The other bands were ignored as being either nonspecific or heteroduplexes. The calculated ratios between out- and in-amplicons are written below each lane. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179165#pone.0179165.s001" target="_blank">S1 Fig</a> for quantification of signals. WT or W: wild type, H: heterozygous, KO or K: knockout (mutant).</p

Accuracy of cbPCR to detect mutants.

<p>(A) Strategy to compare the efficacy of mutant detection by cbPCR. (B) Survival of different clones after HPRT1 editing and 6-TG selection. Viable cells were stained with crystal violet. Clone 7 is absent due to a growth arrest before experiments. (C) Result of cbPCR in the clones of (B). The bands of expected sizes are illustrated by arrowheads. (D) Bands of (C) were quantified and ratios between out- and in-amplicons were calculated. Ranking based on the calculated ratios discriminated 6-TG resistant clones, except for the 6-TG sensitive clone #10. (E) Clone 10 had a point mutation. (F) Sanger sequencing of clone 10 revealed that the point mutation was silent. The other allele had a +1 insertion. Mutations are illustrated in magenta.</p

Comparison of mutation type and and cbPCR results.

<p>Comparison of mutation type and and cbPCR results.</p

Optimization of cbPCR.

<p>(A) Nomenclature of the primers based on their orientation, using Sgpl1 locus as an example. The Cas9 cleavage site is zoomed in to show where the inner primers bind. (B) PCR was performed with or without the addition of the indicated inner primer. The ratios between out- and in-amplicons were calculated. The orientation of the inner primer affected the degree of competition between amplicons. See the changes in the 761 bp- or 748 bp-outer amplicons. The same experiment is performed in two different loci, namely Sgpl1 and Sphk1. (C) PCR was performed with the addition of inner primers of different lengths. The ratios between out- and in-amplicons were calculated. The length of the inner primer affected the degree of competition between amplicons (as seen by the decrease of the 761 bp-outer amplicon) and the tolerance to the mutations (as seen by the appearance of the ~531 bp-inner amplicon when using mutant DNA). (D) Mutants can be discriminated by cbPCR in most of the targets with a common primer design. Bands with expected sizes are marked with dots. The other bands were ignored as being either nonspecific or heteroduplexes. WT or W: wild type, KO or K: knockout (mutant).</p

Competition between amplicons enables the discrimination of mutant cells.

<p>(A) Design of the PCR primers to test whether Sgpl1-mutant cells can be discriminated by conventional endpoint PCR. The 3' end of the forward primer is zoomed to show its location relative to the Cas9 cleavage site. (B) Conventional endpoint PCR with the primer design shown in (A) was performed using wild type or mutant gDNA. The levels of PCR products did not change depending on genotypes or template DNA amount. (C) Design of competition-based PCR (cbPCR) and nomenclature of primers and amplicons. F-in and R-out are the same primers as forward and reverse primers in (A), respectively. (D) Basis of competition between amplicons that occur during cbPCR. (E) PCR was performed with the indicated primer sets, using gDNA from wild type or Sgpl1-mutant cells. The pattern of competition between cbPCR amplicons differed between wild type and mutant gDNA. (F) In contrast to conventional endpoint PCR, cbPCR enables the discrimination of wild type and mutant gDNA. The ratios between out- and in-amplicons are indicated. WT: wild type, KO: knockout (mutant).</p