Primers used in this study
. | Primers 5′ to 3′ . | Annealing temperature, °C . | Product size, bp . |
|---|---|---|---|
| PBGD control primers | |||
| Outer | |||
| PBGD_hum-F | acc aag gag ctt gaa cat gc | 55 | 784 |
| PBGD_hum-R | gaa aga caa cag cat cat gag | ||
| Nested | |||
| PBGD-N-F | aac atg ccc tgg aga | 47 | 144 |
| PBGD-N-R | atg agg gtt ttc ccg | ||
| F8 exon 14-15 RT-PCR | |||
| Outer | |||
| F1 | aag ggt gaa ttt aca aag gac gt | 52 | 1867 |
| R1 | tga aag tta cca tga tat tat ctt | ||
| Nested | |||
| F2 | aga tgg ttt ttc caa gca gca | 54 | 1731 |
| R2 | agc cat cag taa att cct gga a | ||
| SNuPE | |||
| SN-F8Ex14-1241 | gta gaa ggt tca tat ga | 28 | |
| SN-F8Ex14-1269 | cac aca gct cat ttc tc | 28 | |
| F8B-specific RT-PCR | |||
| RT | |||
| Ex19-26/4* | — | — | — |
| Outer | |||
| F8B-Ex1-F1 | cgt atg caa atc gag ggt ct | 52 | 770 |
| Ex19-26/3* | — | — | — |
| Nested | |||
| F8B-Ex1-F2 | gat cca aga ccc tgg gaa g | 53 | 657 |
| Ex23/26-R | agt ggc cac cct cag tag ag | ||
| F8 promoter region | |||
| F8P-F1 | gga gtg aca gga ctc gct tt | 58 | 695 |
| F8P-R1 | aat cgc aaa agg cac aga aa | ||
| F8P-F2 | ctc tcc cta ata aac att aac ctg a | 58 | 699 |
| F8P-R2 | cac tgc tgc cag tat gag ga | ||
| F8P-F3 | ggc tgt agg caa gcc att ta | 58 | 688 |
| F8P-R3 | cat cag aat gta gcc atg gtg | ||
| F8P-F4 | gga gcc ctt taa cag tgt gc | 58 | 844 |
| F8P-R4 | aga aat ccc agt tcc caa cc | ||
| F8P-F5 | tga aaa ttt ggt aat ggg gtc t | 58 | 785 |
| F8P-R5 | gga tct gtg ggc ttt gag aa | ||
| F8P-F6 | cct tca aca gaa aat cca gca | 58 | 828 |
| F8P-R6 | gca aca aga gcg aga ctc tg | ||
| F8P-F7 | gta gag gat ctg cgc ggt ag | 58 | 838 |
| F8P-R7 | cct atg gtg cca caa aat cc | ||
| F8P-F8 | gat ggc tct gtt tag caa tgg | 58 | 802 |
| F8P-R8 | cac gct gag ctc tgt gat gt | ||
| F8 3′ untranslated region | |||
| F8-3′ end-F1 | gcg agg cac agg acc tct a | 58 | 481 |
| F8-3′ end-R1 | tgc ttt gtt cca tgc ttg at | ||
| F8-3′ end-F2 | gga gaa acc tgc atg aaa gc | 58 | 596 |
| F8-3′ end-R2 | ttg gcc atc aca aat ttc aa | ||
| F8-3′ end-F3 | tga gaa tta tag atg ggg ttc aag a | 58 | 593 |
| F8-3′ end-R3 | ggc aga tgg aag gag cag ta | ||
| F8-3′ end-F4 | ccg tga ctg aaa act aga gtc c | 58 | 576 |
| F8-3′ end-R4 | tgg ggt taa ctc ctc tac cg | ||
| F8-3′ end-F5 | tcc tgc ttg acc ctt atc tga | 58 | 689 |
| F8-3′ end-R5 | ctc cca aag tgc cag gat ta | ||
| Methylation analysis | |||
| Outer | |||
| Bi-F8-PN-F1: | ttt aaa att aat aat aaa gta gtt g | 48 | 210 |
| Bi-F8-PN-R1: | acc taa cca ccc taa aat atc | ||
| Nested | |||
| Bi-F8-PN-F2: | ggg agg tgt ttt gta tat aga atg a | 57 | 195 |
| Bi-F8-PN-R2: | tct cca ata aaa ctc acc caa cct t | ||
| SNuPE | |||
| SN-F8-P-1: | ata aag aag tta gat | 28 | |
| HUMARA assay | |||
| HUMARA-Nor-F | 6-FAM-tcc aga atc tgt tcc aga gcg tgc | 60 | 231 |
| HUMARA-Nor-R | ctc tac gat ggg ctt ggg gag aac | ||
| F8 mRNA analysis | — | — | — |
. | Primers 5′ to 3′ . | Annealing temperature, °C . | Product size, bp . |
|---|---|---|---|
| PBGD control primers | |||
| Outer | |||
| PBGD_hum-F | acc aag gag ctt gaa cat gc | 55 | 784 |
| PBGD_hum-R | gaa aga caa cag cat cat gag | ||
| Nested | |||
| PBGD-N-F | aac atg ccc tgg aga | 47 | 144 |
| PBGD-N-R | atg agg gtt ttc ccg | ||
| F8 exon 14-15 RT-PCR | |||
| Outer | |||
| F1 | aag ggt gaa ttt aca aag gac gt | 52 | 1867 |
| R1 | tga aag tta cca tga tat tat ctt | ||
| Nested | |||
| F2 | aga tgg ttt ttc caa gca gca | 54 | 1731 |
| R2 | agc cat cag taa att cct gga a | ||
| SNuPE | |||
| SN-F8Ex14-1241 | gta gaa ggt tca tat ga | 28 | |
| SN-F8Ex14-1269 | cac aca gct cat ttc tc | 28 | |
| F8B-specific RT-PCR | |||
| RT | |||
| Ex19-26/4* | — | — | — |
| Outer | |||
| F8B-Ex1-F1 | cgt atg caa atc gag ggt ct | 52 | 770 |
| Ex19-26/3* | — | — | — |
| Nested | |||
| F8B-Ex1-F2 | gat cca aga ccc tgg gaa g | 53 | 657 |
| Ex23/26-R | agt ggc cac cct cag tag ag | ||
| F8 promoter region | |||
| F8P-F1 | gga gtg aca gga ctc gct tt | 58 | 695 |
| F8P-R1 | aat cgc aaa agg cac aga aa | ||
| F8P-F2 | ctc tcc cta ata aac att aac ctg a | 58 | 699 |
| F8P-R2 | cac tgc tgc cag tat gag ga | ||
| F8P-F3 | ggc tgt agg caa gcc att ta | 58 | 688 |
| F8P-R3 | cat cag aat gta gcc atg gtg | ||
| F8P-F4 | gga gcc ctt taa cag tgt gc | 58 | 844 |
| F8P-R4 | aga aat ccc agt tcc caa cc | ||
| F8P-F5 | tga aaa ttt ggt aat ggg gtc t | 58 | 785 |
| F8P-R5 | gga tct gtg ggc ttt gag aa | ||
| F8P-F6 | cct tca aca gaa aat cca gca | 58 | 828 |
| F8P-R6 | gca aca aga gcg aga ctc tg | ||
| F8P-F7 | gta gag gat ctg cgc ggt ag | 58 | 838 |
| F8P-R7 | cct atg gtg cca caa aat cc | ||
| F8P-F8 | gat ggc tct gtt tag caa tgg | 58 | 802 |
| F8P-R8 | cac gct gag ctc tgt gat gt | ||
| F8 3′ untranslated region | |||
| F8-3′ end-F1 | gcg agg cac agg acc tct a | 58 | 481 |
| F8-3′ end-R1 | tgc ttt gtt cca tgc ttg at | ||
| F8-3′ end-F2 | gga gaa acc tgc atg aaa gc | 58 | 596 |
| F8-3′ end-R2 | ttg gcc atc aca aat ttc aa | ||
| F8-3′ end-F3 | tga gaa tta tag atg ggg ttc aag a | 58 | 593 |
| F8-3′ end-R3 | ggc aga tgg aag gag cag ta | ||
| F8-3′ end-F4 | ccg tga ctg aaa act aga gtc c | 58 | 576 |
| F8-3′ end-R4 | tgg ggt taa ctc ctc tac cg | ||
| F8-3′ end-F5 | tcc tgc ttg acc ctt atc tga | 58 | 689 |
| F8-3′ end-R5 | ctc cca aag tgc cag gat ta | ||
| Methylation analysis | |||
| Outer | |||
| Bi-F8-PN-F1: | ttt aaa att aat aat aaa gta gtt g | 48 | 210 |
| Bi-F8-PN-R1: | acc taa cca ccc taa aat atc | ||
| Nested | |||
| Bi-F8-PN-F2: | ggg agg tgt ttt gta tat aga atg a | 57 | 195 |
| Bi-F8-PN-R2: | tct cca ata aaa ctc acc caa cct t | ||
| SNuPE | |||
| SN-F8-P-1: | ata aag aag tta gat | 28 | |
| HUMARA assay | |||
| HUMARA-Nor-F | 6-FAM-tcc aga atc tgt tcc aga gcg tgc | 60 | 231 |
| HUMARA-Nor-R | ctc tac gat ggg ctt ggg gag aac | ||
| F8 mRNA analysis | — | — | — |
PBGD indicates porphobilinogen deaminase gene; SNP, single nucleotide polymorphism; SNuPE, single nucleotide primer extension; SIRPH, SNuPE-Ion Pair Reverse Phase HPLC (high-performance liquid chromatography); and —, as described by El-Maarri et al.5