PCR amplification of the entire ehxA gene (3 kbp) was performed
with 1 μl template DNA and 0.6 μM of primers ehxA ext. F (5′-CAGG
CAATACCATCATGAAC) and ehxARFLP R (5′-TCAGACAGTTGTCGTTAA
AGTTG). Platinum® PCR SuperMix High Fidelity (Invitrogen Life Technologies,
Grand Island, NY) was added to a total of 20 μl. Amplification
was conducted under the following PCR conditions: Initial denaturing
step at 95 °C for 2min, followed by 30 cycles of 95 °C for 45 s (denaturing),
52 °C for 45 s (annealing), and 72 °C for 3.5 min (polymerization)
and a final extension step at 72 °C for 5 min.