Primer sets and restriction enzyme sites used
Kell mutant . | Names and sequences of primers . | Restriction enzyme sites . |
|---|---|---|
| Asp638Ala | 1369F: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI at nt 1406 and NheI at nt 2043 |
| D638AR: 5′-GATGGCTAGCCCCCCAACGGCTGCAGC-3′ | ||
| Asp638Glu | 1369F: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| D638ER: 5′-GATGGCTAGCCCCCCAACCTCTGCAGC-3′ | ||
| Glu634Asp | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| E634DR: 5′GCTAGCCCCCCAACGTCTGCAGCATTGTCTAAGAATGT-3′ | ||
| Glu634Gly | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| E634GR: 5′-GCTAGCCCCCCAACGTCTGCAGCATTCCCTAAGAATGT-3′ | ||
| Tyr588Phe | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y588FR: 5′-CCCAGGCAGTAAGAGCTGGAAGAAGAT-3′ | ||
| Y588FF: 5′-GAGCTGTTGCACATCTTCTTCCAGCTC-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| Tyr588Asp | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y588DR: 5′-GCCCCCAGGCAGTAAGAGCTGGTCGAAGA-3′ | ||
| Y588DF: 5′-CGAGCTGTTGCACATCTTCGACCAGCTC-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| His694Ala | NheF*: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI at nt 2043 and DraIII at nt 2273 |
| H694AR: 5′-GACTCGGAGGTGTGGAGGGCTGGCAGTGTCGTG-3′ | ||
| H694AF: 5′-AGCCCCCAGGACTCTCACGACACTGCCAGCCCTCCA-3′ | ||
| 2316R*: 5′-CCAGAGCTGGCAGCGGCTGGAGGG-3′ | ||
| Arg700Lys | NheF: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI and DraIII |
| R700KR: 5′-GCACCACGTGCACAGCGGAAATACCT | ||
| GGCAAAGGCTGGGGTGCTGCTGAGGGGCCCGTGGACTTTGAGGTG-3′ | ||
| Tyr615Phe | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| Y615FR: 5′-GCTAGCCCCCCAACGTCTGCAGCATTCTCTAAG | ||
| AATGTGAGGGAGTCATTGAAGGAGGTTCTGCTAGGTAATGGAAAGGC | ||
| AGCAAAATGGCGCTTCAG-3′ | ||
| Tyr615Ala | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y615AR; 5′-GCTAGGTAATGGAAAGGCAGCAGCATGGCG-3′ | ||
| Y615AF: 5′-CTGTGCCTGAAGCGCCATGCTGCTGCCT-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| Asp692Ala | NheF*: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI and DraIII |
| D692AR: 5′-GTGTGGAGGGCTGTGAGTGGCGTGAGAG-3′ | ||
| D692AF: 5′-AGCCCCCAGGACTCTCACGCCACTCAC-3′ | ||
| 2316R*: as above |
Kell mutant . | Names and sequences of primers . | Restriction enzyme sites . |
|---|---|---|
| Asp638Ala | 1369F: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI at nt 1406 and NheI at nt 2043 |
| D638AR: 5′-GATGGCTAGCCCCCCAACGGCTGCAGC-3′ | ||
| Asp638Glu | 1369F: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| D638ER: 5′-GATGGCTAGCCCCCCAACCTCTGCAGC-3′ | ||
| Glu634Asp | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| E634DR: 5′GCTAGCCCCCCAACGTCTGCAGCATTGTCTAAGAATGT-3′ | ||
| Glu634Gly | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| E634GR: 5′-GCTAGCCCCCCAACGTCTGCAGCATTCCCTAAGAATGT-3′ | ||
| Tyr588Phe | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y588FR: 5′-CCCAGGCAGTAAGAGCTGGAAGAAGAT-3′ | ||
| Y588FF: 5′-GAGCTGTTGCACATCTTCTTCCAGCTC-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| Tyr588Asp | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y588DR: 5′-GCCCCCAGGCAGTAAGAGCTGGTCGAAGA-3′ | ||
| Y588DF: 5′-CGAGCTGTTGCACATCTTCGACCAGCTC-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| His694Ala | NheF*: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI at nt 2043 and DraIII at nt 2273 |
| H694AR: 5′-GACTCGGAGGTGTGGAGGGCTGGCAGTGTCGTG-3′ | ||
| H694AF: 5′-AGCCCCCAGGACTCTCACGACACTGCCAGCCCTCCA-3′ | ||
| 2316R*: 5′-CCAGAGCTGGCAGCGGCTGGAGGG-3′ | ||
| Arg700Lys | NheF: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI and DraIII |
| R700KR: 5′-GCACCACGTGCACAGCGGAAATACCT | ||
| GGCAAAGGCTGGGGTGCTGCTGAGGGGCCCGTGGACTTTGAGGTG-3′ | ||
| Tyr615Phe | 1224F: 5′-CTGCAGAGCCACATGATCTTAGGG-3′ | StuI and NheI |
| Y615FR: 5′-GCTAGCCCCCCAACGTCTGCAGCATTCTCTAAG | ||
| AATGTGAGGGAGTCATTGAAGGAGGTTCTGCTAGGTAATGGAAAGGC | ||
| AGCAAAATGGCGCTTCAG-3′ | ||
| Tyr615Ala | 1369F*: 5′-TTCTTCGAGCCCACGCTGGCGGCT-3′ | StuI and NheI |
| Y615AR; 5′-GCTAGGTAATGGAAAGGCAGCAGCATGGCG-3′ | ||
| Y615AF: 5′-CTGTGCCTGAAGCGCCATGCTGCTGCCT-3′ | ||
| NheR*: 5′-TGGTGCCGTAACAGCCTCTTGCTGTATGC-3′ | ||
| Asp692Ala | NheF*: 5′-GCAGACGTTGGGGGGCTAGCCATC-3′ | NheI and DraIII |
| D692AR: 5′-GTGTGGAGGGCTGTGAGTGGCGTGAGAG-3′ | ||
| D692AF: 5′-AGCCCCCAGGACTCTCACGCCACTCAC-3′ | ||
| 2316R*: as above |
The mutated bases are underlined.
Primers used to link the overlapping PCR products.