Figure 5. Strategy to genotype hybrid-derived templates.

The graph underneath each gel print provides an overview of the hybrid tandem, the long-range PCR fragment(s) present in each genotyping reaction and the sequence variation interrogated. For RFLP genotyping, a PCR fragment was amplified, cut with a restriction enzyme and resulting fragments resolved by agarose gel electrophoresis. Fragment lengths are given in bp to the right, marker sizes on the left. (A) Genotyping assay detecting the T-insertion in exon 1 on fragment H. DNA samples with hybrid tandems, CYP2D7 only (control plasmid) and a single hybrid (CYP2D6*66) were completely cut, indicating the presence of the T-insertion. The CYP2D6 control plasmid remained uncut due to the absence of the insertion. (B) Genotyping assay detecting the exon 9 conversion on fragment H. DNA samples with hybrid tandems, a single hybrid (CYP2D6*66) and CYP2D6 only (plasmid control) were cut, indicating that exon 9 is derived from CYP2D6. The CYP2D7 plasmid control remained uncut due to the presence of the exon 9 conversion. (C) Assay as in (A), but performed on triplex long-range PCR products. Since the primers will bind to fragments A (from both alleles) and fragment H (from the tandem hybrid) the resulting band pattern appears heterozygous. A similar (heterozygous) result is obtained when genotyping is performed for the exon 9 conversion (not shown).