Original article - Blood Transfusion

G Fratellanza et al.Table I - Sequences of the specific primers used for PCR and their genomic position on DNAPrimerSequence DNAHPA-1(s) 5’-CTGCAGGAGGTAGAGAGTCGCCATAG-3’ 12447HPA-1(a) 5’-GTGCAATCCTCTGGGACTGACTTG-3’ 12923HPA-2(s) 5’-CCTTCAACCGGCTGACCTCGCTGCC-3’ 3083HPA-2(a) 5’-TTCAGCATTGTCCTGCAGCCAGC-3’ 3685HPA-3(s) 5’-CTCAAGGTAAGAGCTGTGAAGAAGAC-3’ 13834HPA-3(a) 5’-CTCACTACGAGAAGGATCCTGAACCTC-3’ 14281HPA-4(s) 5’-TACCAAGCTGGCCACCCAGATT-3’ 13842HPA-4(a) 5’-CCAAACAGGGAGACCAAGGTAGAA-3’ 14047HPA-5(s) 5’-GTGACCTAAAGAAAGAGG-3’ 1601HPA-5(a) 5’-CTCTCATGGAAAATGGCAG-3’ 1874HPA- 6 (s) 5’- CTGGCTGGCTGGGATCCCAGTG- 3’HPA- 6 (a) 5’- CCCTGCAGTTCTCCTCACCTGAG- 3’Table II - PCR protocol: master mixMaster mix HPA-1 HPA-2 HPA-3 HPA-4 HPA-5 HPA-6Buffer 2µL 2µL 2µL 2µL 2µL 2µLW1(5%) 1µL 1µL 1µL 1µL 1µL 1µLDNTPs (10µM) 0.8µL 0.8µL 0.8µL 0.8µL 0.8µL 0.8µLForward 1µL 1µL 1µL 1µL 0.8µL 0.8µLReverse 1µL 1µL 1µL 1µL 0.8µL 0.8µLMgCl 20.6µL 0.4µL 0.4µL 0.4µL 0.4µL 0.4µLTAQ 0.08µL 0.08µL 0.08µL 0.08µL 0.08µL 0.08µLH 20 13.52µL 13.72µL 13.72µL 13.72µL 14.12µL 14.12µLTable III - PCR protocol: amplification cycles (temperature, time, number of cycles)Cycles HPA-1 HPA-2 HPA-3 HPA-4 HPA-5 HPA-6Denaturation 95 °C x 5’(1) 95 °C x 5’(1) 95 °C x 5’(1) 95 °C x 5’(1) 95 °C x 5’(1) 95 °C x 5’(1)Denaturation 95 °C x 30’’(30) 95 °C x 1’(35) 95 °C x 1’(35) 95 °C x 1’(30) 95 °C x 55’’(36) 95 °C x 1’ (28)Annealing 62 °C x 15’’(30) 68 °C x 1’(35) 60 °C x 1’(35) 60 °C x 30’’(30) 51 °C x 55’’(36) 70 °C x 1’ (28)Extension 72 °C x120’’(30) 72 °C x 1’(35) 72 °C x 1’(35) 72 °C x 30’’(30) 72 °C x 30’’(36) 72 °C x 1’ (28)End cycle 72 °C x5’ 72 °C x5’ 72 °C x5’ 72 °C x 5’ 72 °C x 5’ 72 °C x 5’Table IV - Restriction-enzymes used to digest the amplification products and restriction sequencesAlloantigens Genes Site on cDNA Aa. Restriction sequences Enzyme Undigested amplificate Alleles (bp)HPA-1 a GPIIIa 196: CTG 33:Leu CTGGG Nci I 247 247HPA-1 b CCG Pro CCGGG 77/170HPA-2 a GPIb 524: ACG 145:Thr GACGTC BsaH I 353 111/242HPA-2 b ATG Met GATGTC 353HPA-3 a GPIIb 2622:ATC 843:Ile CATCC Fok I 253 126/127HPA-3 b AGC Ser CAGCC 253HPA-4 a GPIIIa 526: CGA 143:Arg TCGA Taq I 276 22/92/162HPA-4 b CAA Gln TCAA 114/162HPA-5 a GPIa 1648:GAG 505:Glu GAGG Mnl I 274 8/33/136/97HPA-5 b AAG Lys AAGG 8/169/97HPA-6 a GPIIIa 1564: CAG 489: Gln CGGA Mwa I 240 240HPA-6 b CGG Arg CAGG 75/165224Blood Transfus 2005; 3: 222-30