Two technical procedures for specific detection of theRHD deletion in the common RHD⁻haplotypes.

(A) A long-range PCR amplification with primers located in non-Rhesus box sequences and (B) PCR-RFLP with primers located in the Rhesus boxes are shown. The deduced genotypes are indicated. The primers of the long-range PCR were located 5′ of the upstream Rhesus box (primer rez4) and in SMP1exon 1 (primer sr9). RHD⁻ haplotypes were detected specifically (panel A, lanes 1-6). DNA homozygous for theRHD gene was negative because PCR cannot amplify the 70 000-bp DNA stretch of the RHD gene. For the PCR-RFLP method, the PCR amplicons (primer rez7 and rnb31) were digested withPstI. In D-negative haplotypes, there are 3PstI sites in the amplicon (Figure 5) resulting in fragments of 1888 bp, 564 bp, 397 bp, and 179 bp (lanes 1-3). The downstream Rhesus box of D-positive haplotypes lacks 1PstI site, resulting in fragments of 1888 bp, 744 bp, and 397 bp (lanes 7-9).RHD⁺/RHD⁻ heterozygotes show both fragments of 744 bp and 564 bp (lanes 4-6). The 564-bp fragment appears weaker because heterodimers are not cut by PstI. Primer rnb31 does not amplify the upstream Rhesus box of D-positive haplotypes.