Table 2A.

Polymerase chain reaction first-step amplification conditions (Method 1.1 & 1.2)

	First-step amplification
	1	2	3	4	5	6	7	8	9	10
Buffer	2.5a	2.5b	2.5a	2.5a	2.5a	2.5c	2.5a	2.5a	2.5b	2.5d
DNA (ng)	50	50	50	50	50	50	50	50	50	75
Primers (each) (nM)	62.5	125	62.5	125	125	100	125	125	62.5	250
DMSO	–	–	–	–	–	–	–	–	–	2%
dNTPs (uM)	200	200	200	200	200	125	200	200	200	300
MgCl2 (mM)	1.3	2.0	1.5	1.5	1.5	–	2.5	1.5	1.7	–
Taq (units)	0.75Ɨ	0.75Ɨ	1.25Ɨ	0.75Ɨ	1.00Ɨ	0.5uL£	1.25Ɨ	1.00Ɨ	1.25Ɨ	1.25Ŧ
First denaturation (°C:sec)	95:60	95:60	95:60	95:60	95:60	95:120	95:60	95:60	95:60	94:30
[i]Denaturation (°C:sec)	95:20	95:15	95:15	95:15	95:15	95:20	95:15	95:15	95:15	94:20
[i]Annealing(°C:sec)	55:30	60:30	58:30	60:30	50:30	50:20	52:20	50:30	53:30	54:20
[i]Extension(°C:sec)	72:60	72:120	72:120	72:210	72:210	72:60	72:180	72:180	72:180	68:420
[ii]Denaturation (°C:sec)	–	–	–	–	–	–	–	–	–	94:20
[ii]Annealing(°C:sec)	–	–	–	–	–	–	–	–	–	54:20
[ii]Extension(°C:sec)	–	–	–	–	–	–	–	–	–	68:420e
Number of cycles	35	36	30	35	40	30	35	40	35	–g
Last extension(°C:min)	72:7	72:7	72:7	72:7	72:7	72:3	72:7	72:7	72:7	68:10

1: First amplification for CYP2A6*9, *31

2: First amplification fo CYP2A6*2, *24, *25, *26

3: First amplification fo CYP2A6*20, *23, *27

4: First amplification fo CYP2A6*12, *34

5: First amplification fo CYP2A6*1B, *17, *28, *35

6: First amplification fo CYP2A6*4H

7: First amplification fo CYP2A6*4B

8: First amplification fo CYP2A6*7,*8,*10

9: First amplification fo CYP2A6*1X2A

10: First amplification fo CYP2A6*1X2B

^ƗTaq DNA Polymerase (Thermo Scientific)

^£Pfu: PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies)

^ŦLong PCR Enzyme Mix (Fermentas, Canada)

^a10X Taq Buffer with MgCl₂ (Thermo, Canada)

^b10X Taq Buffer with (NH₄)₂SO₄ (Thermo, Canada)

^c10X Pfu Buffer (Agilent Technologies)

^d10X Long PCR Buffer with MgCl₂ (Fermentas, Canada)

^e+5 seconds per cycle

^gTen cycles for [i], 20cycles for [ii]