Table 2B.
Polymerase chain reaction second-step amplification conditions (Method 1.1 & 1.2)
| Second-step amplification
| ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CYP2A6*9 | CYP2A6*31 | CYP2A6*2 | CYP2A6*24 | CYP2A6*25 | CYP2A6*26 | CYP2A6*20 | CYP2A6*23 | CYP2A6*27 | CYP2A6*12 | CYP2A6*34 | CYP2A6*1B | CYP2A6*17 | CYP2A6*28 | CYP2A6*35 | CYP2A6*4H | CYP2A6*4B | CYP2A6*7 | CYP2A6*8 | CYP2A6*10 | CYP2A6*1x2A | CYP2A6*1x2B | |
| Buffer | 2.5b | 2.5b | 2.5a | 2.5a | 2.5a | 2.5a | 2.5a | 2.5a | 2.5a | 2.5a | 2.5a | 2.5b | 2.5b | 2.5a | 2.5a | 2.5b | 2.5a | 2.5a | 2.5b | 2.5b | 2.5b | 2.5c |
| First-step product (μL) | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 | 0.8 |
| Primers (each) (nM) | 62.5 | 62.5 | 125 | 75 | 100 | 62.5 | 125 | 150 | 125 | 125 | 62.5 | 125 | 250 | 125 | 125 | 125 | 125 | 250 | 150 | 100 | 125 | 250 |
| DMSO | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 2% |
| dNTPs (μM) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 300 |
| MgCl2 (mM) | 1.10 | 1.10 | 1.25 | 1.25 | 0.75 | 1.20 | 1.50 | 1.00 | 1.30 | 1.50 | 1.50 | 1.50 | 1.00 | 1.50 | 1.25 | 1.50 | 1.50 | 1.10 | 1.20 | 1.30 | 1.60 | – |
| Taq (units) | 0.4Ɨ | 0.4Ɨ | 0.25Ɨ | 0.3Ɨ | 0.3Ɨ | 0.4Ɨ | 0.3Ɨ | 0.4Ɨ | 0.4Ɨ | 0.5Ɨ | 0.5Ɨ | 0.5Ɨ | 0.5Ɨ | 0.3Ɨ | 0.5Ɨ | 0.5Ɨ | 0.5Ɨ | 0.3Ɨ | 0.4Ɨ | 0.3Ɨ | 0.75Ɨ | 1.25Ŧ |
|
| ||||||||||||||||||||||
| First denaturation (°C:sec) | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 95:60 | 94:30:00 |
| [i]Denaturation (°C:sec) | 95:20 | 95:20 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 95:15 | 94:20 |
| [i]Annealing(°C:sec) | 66:40 | 65:30 | 50:20 | 58:10 | 65:10 | 59:20 | 56:20 | 62:10 | 56:20 | 62:20 | 59:20 | 52:30 | 58:30 | 58:20 | 55:40 | 52:30 | 50:20 | 59:20 | 57:20 | 57:30 | 60:30 | 54:20 |
| [i]Extension(°C:sec) | 72:60 | 72:60 | 72:45 | 72:90 | 72:45 | 72:60 | 72:30 | 72:30 | 72:40 | 72:120 | 72:60 | 72:120 | 72:60 | 72:60 | 72:60 | 72:180 | 72:90 | 72:60 | 72:60 | 72:30 | 72:150 | 68:420 |
| [ii]Denaturation (°C:sec) | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 94:20 |
| [ii]Annealing(°C:sec) | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 54:20 |
| [ii]Extension(°C:sec) | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 68:420d |
| Number of cycles | 20 | 20 | 22 | 24 | 16 | 18 | 18 | 23 | 18 | 25 | 22 | 20 | 25 | 20 | 20 | 25 | 20 | 30 | 20 | 30 | 25 | –e |
| Last extension(°C:min) | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 68:10 | |
Ɨ
Taq DNA Polymerase (Thermo Scientific)
Ŧ
Long PCR Enzyme Mix (Fermentas, Canada)
a
10X Taq Buffer with MgCl2 (Thermo, Canada)
b
10X Taq Buffer with (NH4)2SO4 (Thermo, Canada)
c
10X Long PCR Buffer with MgCl2 (Fermentas, Canada)
d
+5 seconds per cycle
e
Ten cycles for [i], 20cycles for [ii]