Figure 2. Assaying NAHR using real-time PCR.
Panel A shows two duplicated sequences that undergo NAHR as grey and black arrows. Circles above these arrows present variant sites that distinguish these two highly similar sequences, and flank a NAHR hotspot. Repeat-specific PCR primers target these sites in a nested fashion to amplify haplotypes of these sites that are diagnostic of deletions and duplications. The haplotypes diagnostic of deletions are different from those diagnostic of duplications. Panel B shows the results of the second, real-time PCR reaction for each rearrangement-specific haplotype in both blood and sperm-derived DNA. Each plot shows how the relative fluorescence intensity (Δ) increases with cycle number. Each line represents a single PCR reaction on an independent pool of genomes. It is expected that only a minority of reactions will contain a rearrangement breakpoint. Red lines indicate pools of genomes that contain a rearrangement breakpoint (positive), blue lines indicate reactions that do not contain a breakpoint (negative). The discrete clustering of these two classes of reaction are readily apparent in each of the eight assays. The presence of amplified DNA in negative reactions results from stochastic base mis-incorporation during linear amplification of non-recombinant haplotypes that generates a synthetic recombinant haplotype at some point during the thermocycling.