Background
Methods
Blood samples
Serological studies
Molecular studies
Genomic DNA extraction
Amplification of RHDexons
Primer name* | Sequence 5′ to 3′ | Location#
| Specificity | Product size (bp) |
---|---|---|---|---|
E1-s | ATGCCTGGTGCTGGTGGA | promoter, -43 to -26 |
RHD/CE
| 228 |
E1-a | ATTTGCTCCTGTGACCACTT | intron 1, 37 to 18 |
RHD
| |
E1-seq | ATGCCTGGTGCTGGTGGA | |||
E2-s | TGACGAGTGAAACTCTATCTCGAT | intron 1, -1064 to -1041 |
RHD
| 1475 |
E2-a | GGATTCCTTGTGATACACGGAGTAG | intron 2, 224 to 200 |
RHD
| |
E2-seq | TGACGAGTGAAACTCTATCTCGAT | |||
E3-s | GTCGTCCTGGCTCTCCCTCTCT | intron 2, -29 to -8 |
RHD
| 219 |
E3-a | CTTTTCTCCCAGGTCCCTCCT | intron 3, 39 to 19 |
RHD/CE
| |
E3-seq | GTCGTCCTGGCTCTCCCTCTCT | |||
E4-s | GCCGACACTCACTGCTCTTAC | intron 3, -36 to -16 |
RHD/CE
| 378 |
E4-a | TGAACCTGCTCTGTGAAGTGC | intron 4, 194 to 174 |
RHD
| |
E4-seq | GCCGACACTCACTGCTCTTAC | |||
E5-s | TACCTTTGAATTAAGCACTTCACAG | intron 4, -267 to -233 |
RHD
| 1458 |
E5-a | TTATTGGCTACTTGGTGCC | intron 5, 1024 to 1006 |
RHD
| |
E5-seq | TACCTTTGAATTAAGCACTTCACAG | |||
E6-s | CAGGGTTGCCTTGTTCCCA | intron 5, -95 to -77 |
RHD/CE
| 274 |
E6-a | CTTCAGCCAAAGCAGAGGAGG | intron 6, 41 to 21 |
RHD
| |
E6-seq | CAGGGTTGCCTTGTTCCCA | |||
E7-s | TGCCCATCCCCCTTTGGTGGCC | intron 6, -106 to -85 |
RHD
| 411 |
E7-a | CCAAGGTAGGGGCTGGACAG | intron 7, 171 to 152 |
RHD
| |
E7-seq | TGCCCATCCCCCTTTGGTGGCC | |||
E8-s | GGTCAGGAGTTCGAGATCAC | intron 7, -594 to -575 |
RHD
| 709 |
E8-a | TGGCAATGGTGGAAGAAAG | intron 8, 35 to 16 |
RHD/CE
| |
E8-seq | GGTCAGGAGTTCGAGATCAC | |||
E9-s | CTGTCGTTTTGACACACAATATTTC | intron 8, -91 to -67 |
RHD
| 190 |
E9-a | CACGTTAATAGGTGAAAAATCTTACC | intron 9, 25 to exon 9, 1227 |
RHD
| |
E9-seq | CTGTCGTTTTGACACACAATATTTC | |||
E10-s | CAAGAGATCAAGCCAAAATCAGT | intron 9, -67 to -45 |
RHD/CE
| 382 |
E10-a | AGCTTACTGGATGACCACCA | 3’UTR, 291 to 272 |
RHD
| |
E10-seq | CAAGAGATCAAGCCAAAATCAGT | |||
RHD1227A-s | GATGACCAAGTTTTCTGGAAA | exon 9, 1207 to 1227 |
RHD1227A | 109 |
RHD1227A-a | CATAAACAGCAAGTCAACATATATACT | intron9, 88 to 62 |
RHD
| |
RHD1227A-seq | GATGACCAAGTTTTCTGGAAA | |||
β-actin-s | GGAAATCGTGCGTGACATT | — | 473 | |
β-actin-a | CGTCATACTCCTGCTTGCTG | — |
PCR-restriction fragment length polymorphism (PCR-RFLP) for RHDzygosity determination
Purification and sequencing
Results
Serologic studies
Sample no. | Number of subjects | RhD | RhCcEe |
RHD zygosity* |
RHD allele | ||
---|---|---|---|---|---|---|---|
Micro-plate | IAT | Adsorption/elute | |||||
13001-13030 | 30 | – | – | + | Ccee |
RHD+/RHD-
|
RHD1227A |
13031-13033 | 3 | – | – | + | CCee |
RHD+/RHD-
|
RHD1227A |
13034-13035 | 2 | – | – | + | Ccee |
RHD+/RHD+
|
RHD1227A |
13036 | 1 | – | – | + | CCEe |
RHD+/RHD-
|
RHD1227A |
13037 | 1 | – | – | + | CcEe |
RHD+/RHD-
|
RHD1227A |
13038 | 1 | – | – | + | Ccee |
RHD+/RHD-
|
RHD-CE (2-5)-D |
13039 | 1 | – | – | + | Ccee |
RHD+/RHD-
|
RHD-CE (4-7)-D |
13040 | 1 | – | – | + | Ccee |
RHD+/RHD+
|
RHD93T>A |
13041 | 1 | – | – | + | Ccee |
RHD+/RHD-
|
RHD838G>A |