Figure 2

The novel REMS-qPCR improves the quantitative detection limit for low-abundant point mutations. This method requires pre-PCR cleavage of the high-abundant variant (non-target DNA here: recipient-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10³. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).