Primer sequences and PCR conditions for GPIbα amplifications
Region amplified . | Size, bp . | Primer sequence . | PCR conditions . |
|---|---|---|---|
| Fragment I | 976 | 5′–CCAGGGGATGCAGGGGGATCC–3′ (3019–3039) | Denaturation 94°C, 5 min |
| 5′–CTTGTGGCACGCACCTTATC–3′ (3975–3994) | Annealing 52°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Fragment II | 1160 | 5′–GTGATGAAGGTGACACAGACC–3′ (3919–3939) | Denaturation 94°C, 5 min |
| 5′–GACAGGGCTTCTGATACAGAA–3′ (5066–5086) | Annealing 60°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Macroglycopeptide region | 320 or 359 | 5′–CCATCACATTCTCCAAAACTCC–3′ (4174–4195) | Denaturation 94°C, 5 min |
| 5′–TGGCTGATCAAGTTCAGGGATG–3′ (4472–4493) | Annealing 57°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Entire GPIbα-coding region | 1882 | 5′–ACTAGTATGCCTCTCCTCCTCTTGCTG–3′ | Denaturation 92°C, 30 s |
| 5′–TCAGAGGCTGTGGCCAGAGTA–3′ | Annealing 61°C, 30 s | ||
| Extension 68°C, 20 min |
Region amplified . | Size, bp . | Primer sequence . | PCR conditions . |
|---|---|---|---|
| Fragment I | 976 | 5′–CCAGGGGATGCAGGGGGATCC–3′ (3019–3039) | Denaturation 94°C, 5 min |
| 5′–CTTGTGGCACGCACCTTATC–3′ (3975–3994) | Annealing 52°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Fragment II | 1160 | 5′–GTGATGAAGGTGACACAGACC–3′ (3919–3939) | Denaturation 94°C, 5 min |
| 5′–GACAGGGCTTCTGATACAGAA–3′ (5066–5086) | Annealing 60°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Macroglycopeptide region | 320 or 359 | 5′–CCATCACATTCTCCAAAACTCC–3′ (4174–4195) | Denaturation 94°C, 5 min |
| 5′–TGGCTGATCAAGTTCAGGGATG–3′ (4472–4493) | Annealing 57°C, 1 min | ||
| Extension 72°C, 5 min | |||
| Entire GPIbα-coding region | 1882 | 5′–ACTAGTATGCCTCTCCTCCTCTTGCTG–3′ | Denaturation 92°C, 30 s |
| 5′–TCAGAGGCTGTGGCCAGAGTA–3′ | Annealing 61°C, 30 s | ||
| Extension 68°C, 20 min |
The underlined sequence represents a SpeI restriction site generated to facilitate cloning.