Sequencing Method for all 10 RHD Exons From Genomic DNA
| RHD Exon . | PCR Primers . | RHD-Specific* . | PCR Conditions† . | Sequencing Primers . | RHD-Specific* . | ||
|---|---|---|---|---|---|---|---|
| Sense . | Antisense . | Extension . | Annealing . | ||||
| Exon 1 | rb13 | rb11d | No | 10 min | 60°C | re01 | Yes |
| Exon 2 | re12d | re23 | Yes | 3 min | 65°C | re13 | No |
| Exon 3 | ra21 | rb21 | No | 10 min | 60°C | re31 and rb20d | Yes |
| Exon 4 | rb46 | rb12 | Yes | 10 min | 60°C | rb22 | No |
| Exon 5 | rb11 | rh2 | Yes | 10 min | 60°C | rb24 | No |
| Exon 6 | rf51 | re71 | Yes | 10 min | 60°C | rb25 | No |
| Exon 7 | re617 | rb44 | No | 10 min | 60°C | re621 and re75 | Yes |
| Exon 8 | rb52 | rb93 | Yes | 10 min | 60°C | re73 | No |
| Exon 9 | rb52 | rb93 | Yes | 10 min | 60°C | re82/re83 | Yes/no |
| Exon 10 | re91 | rr4 | Yes | 10 min | 60°C | rr3/rh7 | Yes/no |
| RHD Exon . | PCR Primers . | RHD-Specific* . | PCR Conditions† . | Sequencing Primers . | RHD-Specific* . | ||
|---|---|---|---|---|---|---|---|
| Sense . | Antisense . | Extension . | Annealing . | ||||
| Exon 1 | rb13 | rb11d | No | 10 min | 60°C | re01 | Yes |
| Exon 2 | re12d | re23 | Yes | 3 min | 65°C | re13 | No |
| Exon 3 | ra21 | rb21 | No | 10 min | 60°C | re31 and rb20d | Yes |
| Exon 4 | rb46 | rb12 | Yes | 10 min | 60°C | rb22 | No |
| Exon 5 | rb11 | rh2 | Yes | 10 min | 60°C | rb24 | No |
| Exon 6 | rf51 | re71 | Yes | 10 min | 60°C | rb25 | No |
| Exon 7 | re617 | rb44 | No | 10 min | 60°C | re621 and re75 | Yes |
| Exon 8 | rb52 | rb93 | Yes | 10 min | 60°C | re73 | No |
| Exon 9 | rb52 | rb93 | Yes | 10 min | 60°C | re82/re83 | Yes/no |
| Exon 10 | re91 | rr4 | Yes | 10 min | 60°C | rr3/rh7 | Yes/no |
To achieve RHD specificity for genomic nucleotide sequencing, the PCR primer pairs or the sequencing primer or both must not concurrently detect RHCE-derived nucleotide sequences. Primer sequences are given in Table 1.
Primers were used at a concentration of 1 nmol/L in the Expand High Fidelity PCR System (Boehringer Mannheim, Mannheim, Germany). In the exon 10 PCR, the concentration of MgCl2 was 2.0 nmol/L. Denaturation was 20 seconds at 92°C, annealing 30 seconds, elongation temperature 68°C. Elongation time was increased by 20 seconds for each cycle after the 10th cycle, except for the re12d/re23 primer pair.