Figure 5

RHD PCR-SSP optimized for specificity. The PCR is performed as a modular system consisting of two multiplex reactions. An RHD intron 4/exon 7 multiplex PCR-SSP (Panel A) is combined with an RHD intron 7 PCR that is multiplexed with reactions for the specific detection of RHD(W16X) and RHDψ (Panel B). Results are shown for a normal D positive sample (lane 1), a normal D negative sample (lane 2), several rare D negative samples (lanes 3 to 6) and major D positive RHD variants (lanes 7 and 8). Standard D positive and D negative samples and D categories VI and IV are recognized in panel A. RHD-CE(8-9)-D is detected in panel B by the absence of the intron 7 band (lane 3). The presence of RHD(W16X) and RHDψ is detected in panel B because of their specific amplicons (lanes 4 and 5). Amplicon size is Panel A, control, 434 bp (HGH gene); intron 4, 226 bp; exon 7, 123 bp; Panel B, control, 659 bp (chromosome 1 genomic sequence about 90,000 bp 5' of Rhesus box); intron 7, 390 bp; RHD(W16X), 248 bp; RHDψ, 154 bp. The internal control amplicons, which were devised to be larger than the specific amplicons, may be suppressed because of competition, if a specific product is amplified.