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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Pharmacogenomics. 2015 Dec 15;17(2):147–162. doi: 10.2217/pgs.15.156

Table 4A.

Polymerase chain reaction first-step amplification conditions (Methods 2.1 & 2.2)

First-step amplification
I II *12, *34
Buffer 1xPfu buffer 1xPfu buffer 1X Taq Buffer with MgCl2
DNA (ng) 50 50 50
Primers (each) (nM) 100 100 125
dNTPs (uM) 62.5 62.5 200
MgCl2 (mM) na na 1.5
TaqƗ 0.5uL to 25ulƗ 0.5uL to 25ulƗ 0.75U to 25ul £
First denaturation (°C:sec) 95:120 95:120 95:60
Denaturation (°C:sec) 95:20 95:20 95:15
Annealing(°C:sec) 60:15 55:30 60:30
Extension(°C:sec) 72:75 72:60 72:210
Number of cycles 40 40 35
Last extension(°C:sec) 72:180 72:180 72:7

I: First amplification for 1st region (CYP2A6*9, *20, *23, *24, *31)

II: First amplification for 2nd region (CYP2A6*17, *35, *1B)

Ɨ

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

£

Taq DNA Polymerase (Thermo Scientific)