DNA Extraction and Quantification of amoA Gene Abundance

Soil DNA was extracted with the Qiagen DNeasy PowerSoil Kit (Qiagen, Germantown, MD) using 0.30 g field-moist soil. The abundance of soil AOB and AOA was quantified by targeting the amoA gene, which encodes the alpha subunit of AMO, with primer amoA-1F/amoA-2R (Rotthauwe et al., 1997) and Arch-amoAF/Arch-amoAR (Francis et al., 2005), respectively. Quantitative PCR was performed on QuantStudio 7 Flex (Genomic Core, Michigan State University) with Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA). The 25 μl reaction mixture including 12.5 μl Power SYBR Green Master Mix, 1.25 μl forward and reverse primers (10 μM), 9 μl molecular grade water, and 1 μl soil DNA extract (10-fold diluted) was first mixed in 96-well plates and then 20 μl aliquots were transferred to a 384-well plate before analysis. The thermal cycling conditions for quantitative PCR were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 60°C (AOB) or 58°C (AOA) for 1 min and a final extension at 72°C for 45 s. Fluorescence intensity was measured during the 72°C step of each cycle. Standard curves were constructed with plasmids containing cloned amoA products from soil DNA from the same site. Amplification efficiencies ranged between 76 and 98%, with standard curve R² for both AOA and AOB > 0.99.