PCR primers
| Primers/primer ID . | Sequence 5′ → 3′ . | Location . | Annealing temperature, °C . | Product size, bp . |
|---|---|---|---|---|
| Primers used for cloning and sequencing of exon 2 | ||||
| S-2A | TGA GCT GAG ATC CTG TTT TAC TCT | Intron 1 | 55 | 474 |
| AS-2A | TGA ATG GAT TAT CGT GAC TCT CTC | Exon 2 | 55 | |
| S-2B | TGC CAT TGC TCA GGT ACA TAT TT | Exon 2 | 55 | 445 |
| AS-2B | TCA ACA GCT TTC TAT AGG GAA AAA | Intron 2 | 55 | |
| Primers used for PCR, SSCP, and direct sequencing of exons 2 to 6 | ||||
| S-21 | GTGTTTTTGTTTCTGAGCTGAGAT | Intron 1 | 60 | 251 |
| AS-22 | CGCCTCCCATATTTGGGTAG | Exon 2 | 60 | |
| S-23 | AGAACCCGTACCCATAATATATG | Exon 2 | 60 | 311 |
| AS-11 | GAAAAAGAACTATGTGAATGGAT | Exon 2 | 60 | |
| S-24 | GCTCAGGTACATATTTGTTCGGG | Exon 2 | 60 | 269 |
| AS-25 | TTTCAGGATTCAGTGCTGCTC | Exon 2 | 60 | |
| S-26 | TGTGATACAAACCACATCATTTG | Exon 2 | 60 | 274 |
| AS-13 | GCCAAACAATCATTATATACAAG | Intron 2 | 60 | |
| S-17* | TGGATTCTCAGTCGTTCTGGTGA | Intron 2 | 60 | 244 |
| AS-18 | CTTCTCCCTCAAGACAACATGAA | Intron 3 | 60 | |
| S-5 | TCACTCCTTTCTTCCCCTCTC | Intron 3 | 58 | 267 |
| AS-14 | AATCCCAACCATGAATGCCCTC | Intron 4 | 58 | |
| S-15 | TCTTCCTGAGGTATGATTATGGTG | Intron 4 | 57 | 298 |
| AS-16 | AAGAGTTCAGACACAATCTTTTCTC | Intron 5 | 57 | |
| S-19* | GGTCATTGTTATCATGGGACAG | Intron 5 | 58 | 361 |
| AS-20 | TCTTACAATCTAGGCTTCCTTC | Intron 6 | 58 | |
| Primers used for RT-PCR of PIG-A fragments for SSCP | ||||
| S-35 | CTC TAA GAA CTG ATG TCT AAA CCG | NA | NA | 176 |
| As-22 | C GCC TCC CAT ATT TGG GTA G | NA | NA | |
| S-23 | AGA ACC CGT ACC CAT AAT ATA TG | NA | NA | 311 |
| As-11 | GAA AAA GAA CTA TGT GAA TGG AT | NA | NA | |
| S-24 | GCT CAG GTA CAT ATT TGT TCG GG | NA | NA | 269 |
| As-25 | TTT CAG GAT TCA GTG CTG CTC | NA | NA | |
| S-26 | TGT GAT ACA AAC CAC ATC ATT TG | NA | NA | 242 |
| As-36 | TCT GGA TAT TTC TGA CAG AGT TCA G | NA | NA | |
| S-38 | ATT GTT GTT GTC AGC AGA CTT G | NA | NA | 333 |
| AsPIG-Ae | CTC AGG AAT TCC ACC AAC TCT | NA | NA | |
| S-40 | GCA TGG CGA TCG TGG AAG CAG | NA | NA | 293 |
| As-41 | TCG TTT GTC CAT TGG CAA CAC AGC | NA | NA | |
| S-30 | CTG AAG TCA GGG ACA TTG CC | NA | NA | 381 |
| As-20 | TCT TAC AAT CTA GGC TTC CTT C | NA | NA | |
| Primers for 1st and 2nd round mutation-specific PCR | ||||
| SD-1 | GTA AGG AAA ATA CTA AG | Exon 2 | 48 | 188 |
| ASD-1 | AGT CTA CAA TGC AAT TA | Intron 2 | 48 | |
| SD-2 | GTA AGG AAA ATA CTA AG | Exon 2 | 48 | 168 |
| ASD-2 | CTA TCA TTT CAT TAC CT | Intron 2 | 48 |
| Primers/primer ID . | Sequence 5′ → 3′ . | Location . | Annealing temperature, °C . | Product size, bp . |
|---|---|---|---|---|
| Primers used for cloning and sequencing of exon 2 | ||||
| S-2A | TGA GCT GAG ATC CTG TTT TAC TCT | Intron 1 | 55 | 474 |
| AS-2A | TGA ATG GAT TAT CGT GAC TCT CTC | Exon 2 | 55 | |
| S-2B | TGC CAT TGC TCA GGT ACA TAT TT | Exon 2 | 55 | 445 |
| AS-2B | TCA ACA GCT TTC TAT AGG GAA AAA | Intron 2 | 55 | |
| Primers used for PCR, SSCP, and direct sequencing of exons 2 to 6 | ||||
| S-21 | GTGTTTTTGTTTCTGAGCTGAGAT | Intron 1 | 60 | 251 |
| AS-22 | CGCCTCCCATATTTGGGTAG | Exon 2 | 60 | |
| S-23 | AGAACCCGTACCCATAATATATG | Exon 2 | 60 | 311 |
| AS-11 | GAAAAAGAACTATGTGAATGGAT | Exon 2 | 60 | |
| S-24 | GCTCAGGTACATATTTGTTCGGG | Exon 2 | 60 | 269 |
| AS-25 | TTTCAGGATTCAGTGCTGCTC | Exon 2 | 60 | |
| S-26 | TGTGATACAAACCACATCATTTG | Exon 2 | 60 | 274 |
| AS-13 | GCCAAACAATCATTATATACAAG | Intron 2 | 60 | |
| S-17* | TGGATTCTCAGTCGTTCTGGTGA | Intron 2 | 60 | 244 |
| AS-18 | CTTCTCCCTCAAGACAACATGAA | Intron 3 | 60 | |
| S-5 | TCACTCCTTTCTTCCCCTCTC | Intron 3 | 58 | 267 |
| AS-14 | AATCCCAACCATGAATGCCCTC | Intron 4 | 58 | |
| S-15 | TCTTCCTGAGGTATGATTATGGTG | Intron 4 | 57 | 298 |
| AS-16 | AAGAGTTCAGACACAATCTTTTCTC | Intron 5 | 57 | |
| S-19* | GGTCATTGTTATCATGGGACAG | Intron 5 | 58 | 361 |
| AS-20 | TCTTACAATCTAGGCTTCCTTC | Intron 6 | 58 | |
| Primers used for RT-PCR of PIG-A fragments for SSCP | ||||
| S-35 | CTC TAA GAA CTG ATG TCT AAA CCG | NA | NA | 176 |
| As-22 | C GCC TCC CAT ATT TGG GTA G | NA | NA | |
| S-23 | AGA ACC CGT ACC CAT AAT ATA TG | NA | NA | 311 |
| As-11 | GAA AAA GAA CTA TGT GAA TGG AT | NA | NA | |
| S-24 | GCT CAG GTA CAT ATT TGT TCG GG | NA | NA | 269 |
| As-25 | TTT CAG GAT TCA GTG CTG CTC | NA | NA | |
| S-26 | TGT GAT ACA AAC CAC ATC ATT TG | NA | NA | 242 |
| As-36 | TCT GGA TAT TTC TGA CAG AGT TCA G | NA | NA | |
| S-38 | ATT GTT GTT GTC AGC AGA CTT G | NA | NA | 333 |
| AsPIG-Ae | CTC AGG AAT TCC ACC AAC TCT | NA | NA | |
| S-40 | GCA TGG CGA TCG TGG AAG CAG | NA | NA | 293 |
| As-41 | TCG TTT GTC CAT TGG CAA CAC AGC | NA | NA | |
| S-30 | CTG AAG TCA GGG ACA TTG CC | NA | NA | 381 |
| As-20 | TCT TAC AAT CTA GGC TTC CTT C | NA | NA | |
| Primers for 1st and 2nd round mutation-specific PCR | ||||
| SD-1 | GTA AGG AAA ATA CTA AG | Exon 2 | 48 | 188 |
| ASD-1 | AGT CTA CAA TGC AAT TA | Intron 2 | 48 | |
| SD-2 | GTA AGG AAA ATA CTA AG | Exon 2 | 48 | 168 |
| ASD-2 | CTA TCA TTT CAT TAC CT | Intron 2 | 48 |
Primers S17 and S19 are derived from Iida et al.17
NA indicates not applicable.