PCR Amplifications and Direct Sequencing of γ-Glutamyl Carboxylase Gene
| Exon No. . | PCR Conditions . | No. of Cycles . | Primer . | Sequence, 5′ → 3′ . |
|---|---|---|---|---|
| Exon I | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-I-5 | CTA GGG AAG CAA ATT CTC CTG |
| E-I-3.2 | ACC GGG AGA CAC TGG CGT C | |||
| Exon II | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-II-5 | GAG CTG TTG GTG CAG TGA TTT CT |
| E-II-3 | AGA GAT TGT CAT TCT CCA CTC T | |||
| Exon III | 94°C/45 s 56°C/30 s 72°C/1 min | 30 | 323N | GGT TCT TGA TGG TGC TAG |
| E-III-3 | TTC ACC AGC ATG CTT CTA TTT C | |||
| Exon IV | 94°C/45 s 55°C/30 s 72°C/1 min | 30 | 323N | GGT TCT TGA TGG TGC TAG |
| E-IV-3 | TAA CCA TCC TGA CCC AGC CA | |||
| Exon V | 94°C/45 s 49°C/30 s 72°C/1 min | 30 | 630N | CCC CTC AAT GTT TAC CT |
| 350C | GGG TTG AAG GCT ATT CC | |||
| Exon VI | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VI-5 | TGT AAC TCA GGA GCA TGG ATT C |
| E-VI-3 | CAT TAC TGA GAG GAT GCG TCA CCT | |||
| Exon VII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VII-5.2 | TTC GTG CTG TGA TGT GCT TTG A |
| E-VII-3 | TGG CTA GTC CCT TCC TGC AAA ACT G | |||
| Exon VIII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VIII-5 | GAG CCC AGC CAA ACT CCT |
| E-IX-3 | ATG TCT GAT GCT GAC AA | |||
| Exon IX | 94°C/45 s 58°C/30 s 72°C/1 min | 30 | E-IX-5 | CTG GTT TTG CAG CCC CTT CTT |
| 1370C | CCC AGG GTT AAG GTA GCC | |||
| Exon X | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-IX-5 | CTG GTT TTG CAG CCC CTT CTT |
| E-X-3 | AAG CAA GGG CTG TTC ATC TTG G | |||
| Exon XI | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XI-5.4 | GGT GGC TGT GATG TCC TTA GAA |
| E-XI-3.4 | AAG ACA GAA AAG CCT CTC CTC A | |||
| Exon XII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XII-5.4 | GCC ATG GGG TGG GAT GAT GAA C |
| E-XII-3.4 | TGC ACT CAG TTC TTT CTG CTG TTG | |||
| Exon XIII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XIII-5.2 | GGA GGC CAT AAG CTG GCT AGA G |
| E-XIII-3 | GGC TAG AAC ATC ATT CAT AAC C | |||
| Exon XIV | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XIV-5 | CTA GCT GGC AGA AGA GGA GTT C |
| E-XIV-3 | TAT GAT GGC AAT GAC AAA ATA TTG | |||
| Exon XV | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XV-5 | CTA GCT GGC AGA AGA GGA GTT C |
| E-XV-3 | TGT CCA TTG CAT AGA ATG GGT C |
| Exon No. . | PCR Conditions . | No. of Cycles . | Primer . | Sequence, 5′ → 3′ . |
|---|---|---|---|---|
| Exon I | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-I-5 | CTA GGG AAG CAA ATT CTC CTG |
| E-I-3.2 | ACC GGG AGA CAC TGG CGT C | |||
| Exon II | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-II-5 | GAG CTG TTG GTG CAG TGA TTT CT |
| E-II-3 | AGA GAT TGT CAT TCT CCA CTC T | |||
| Exon III | 94°C/45 s 56°C/30 s 72°C/1 min | 30 | 323N | GGT TCT TGA TGG TGC TAG |
| E-III-3 | TTC ACC AGC ATG CTT CTA TTT C | |||
| Exon IV | 94°C/45 s 55°C/30 s 72°C/1 min | 30 | 323N | GGT TCT TGA TGG TGC TAG |
| E-IV-3 | TAA CCA TCC TGA CCC AGC CA | |||
| Exon V | 94°C/45 s 49°C/30 s 72°C/1 min | 30 | 630N | CCC CTC AAT GTT TAC CT |
| 350C | GGG TTG AAG GCT ATT CC | |||
| Exon VI | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VI-5 | TGT AAC TCA GGA GCA TGG ATT C |
| E-VI-3 | CAT TAC TGA GAG GAT GCG TCA CCT | |||
| Exon VII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VII-5.2 | TTC GTG CTG TGA TGT GCT TTG A |
| E-VII-3 | TGG CTA GTC CCT TCC TGC AAA ACT G | |||
| Exon VIII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-VIII-5 | GAG CCC AGC CAA ACT CCT |
| E-IX-3 | ATG TCT GAT GCT GAC AA | |||
| Exon IX | 94°C/45 s 58°C/30 s 72°C/1 min | 30 | E-IX-5 | CTG GTT TTG CAG CCC CTT CTT |
| 1370C | CCC AGG GTT AAG GTA GCC | |||
| Exon X | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-IX-5 | CTG GTT TTG CAG CCC CTT CTT |
| E-X-3 | AAG CAA GGG CTG TTC ATC TTG G | |||
| Exon XI | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XI-5.4 | GGT GGC TGT GATG TCC TTA GAA |
| E-XI-3.4 | AAG ACA GAA AAG CCT CTC CTC A | |||
| Exon XII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XII-5.4 | GCC ATG GGG TGG GAT GAT GAA C |
| E-XII-3.4 | TGC ACT CAG TTC TTT CTG CTG TTG | |||
| Exon XIII | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XIII-5.2 | GGA GGC CAT AAG CTG GCT AGA G |
| E-XIII-3 | GGC TAG AAC ATC ATT CAT AAC C | |||
| Exon XIV | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XIV-5 | CTA GCT GGC AGA AGA GGA GTT C |
| E-XIV-3 | TAT GAT GGC AAT GAC AAA ATA TTG | |||
| Exon XV | 94°C/45 s 57°C/30 s 72°C/1 min | 30 | E-XV-5 | CTA GCT GGC AGA AGA GGA GTT C |
| E-XV-3 | TGT CCA TTG CAT AGA ATG GGT C |