DNA Extraction and Quantitative PCR (qPCR) Amplification of mcrA Gene

Gene copies of methyl coenzyme reductase M α subunit, mcrA, were used to determine the presence of methanogens and further interpret CH₄ production observed in the microcosms. DNA was extracted from subsamples of homogenized frozen bulk soil (pre-incubation) and microcosms incubated at −2, 4, and 8°C (after 30 and either 60 or 75 days of incubation). The total DNA was extracted from 0.25 g of wet soil using the PowerLyzer PowerSoil DNA Isolation Kit (MoBio Laboratories) according to the manufacturer’s protocol. DNA quality were assessed (Nanodrop2000, Thermo Scientific) and concentrations determined by Qubit 3.0 Fluorometer (Life Technologies) using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen). Soil samples containing high concentrations of organic matter and humic acids generally resulted in low purity DNA (A₂₆₀/A₂₈₀ < 1.5). These samples were further purified using the Wizard DNA Clean-Up System (Promega) following the manufacturer’s protocol with sample recovery ranging from 70 to 98% yield. Only samples with high-purity DNA (A₂₆₀/A₂₈₀ > 1.7) were used for downstream applications. Triplicate DNA extractions were performed, and samples were frozen at −20°C until quantitative PCR (qPCR) analysis.

The qPCR was performed with primers mlas (5′ GGT GGT GTM GGD TTC ACM CAR TA) and mcrA-rev (5′ CGT TCA TBG CGT AGT TVG GRT AGT) using the non-specific fluorophore iQ SYBR Green SuperMix (BioRad Laboratories Inc.) (Luton et al., 2002). All qPCR assays were performed using a Bio-Rad iCycler per the method described in Supplementary Table S1. Reaction mixtures contained 19 μL of the master mix and 1 μL of template DNA per reaction. Triplicates of no-template controls, containing diethylpyrocarbonate (DEPC)-treated water were included in each run. In preliminary experiments the annealing temperatures of all reactions were optimized for high-specificity and high yield when amplifying the samples. After each qPCR run, melt curve analysis to verify the presence of the desired amplicon was performed by increasing the temperature from 60°C to 95°C in 0.5°C increments every 5 s.

The concentration of mcrA gene (C_target [copies μL^–1]) in the DNA standard, Methanococcus maripaludis C5 genomic DNA, was calculated from the DNA concentration (C_DNA[ng μL^–1] = 6.1 of the standard), the length of the DNA standard (l_DNA[bp] = 1,661,137), the number of targets per DNA fragment (n_target[copies] = 1 mcrA copy per genome), Avogadro’s number (N_A) (6.022 × 10²³ bp mol^–1), and the average molar mass of a double-stranded base pair (M_bp = 660 g mol^–1) using Eq. 2 (Brankatschk et al., 2012). The C_target value for M. maripaludis C5 was 3.3 × 10⁶ mcrA copies μL^–1.

Two separate strategies were employed to estimate the efficiency (E) of qPCR amplification of soil DNA samples based on the number of cycles necessary to reach the threshold fluorescence values (C_T). These methods estimated either the efficiency from a standard dilution series (E_ds) or the efficiency based on fluorescence increase (E_fi) for each sample, as described below.

The first approach was the frequently used standard curve (SC) method of absolute quantification wherein the linear regression of log(N_{0 standard}) versus C_T gives the intercept a and slope b of the standard curve (Eq. 3). The number of copies in the sample, N_{0 sample}, can be calculated based on the linear regression of a 10-fold dilution series (ds) of the standard. Using this method, the limit of quantitation of mcrA gene concentration was estimated to be 10² copies μL^–1 (C_T = 29, C.V. = 0.011). The slope b of the linear regression (r² > 0.999) was used to estimate the efficiency from dilution series, E_ds (Eq. 4).

Using this method, the PCR amplification efficiency for all runs was determined at 98 – 101% (E_ds = 1.98 to 2.01). This method assumes E_ds of the sample is the same as that of the standard, thus introducing the possibility of increased quantification errors.

Raw fluorescence data were exported from the Bio-Rad iCycler system and imported into the LinRegPCR program (version 2014.1) (Ruijter et al., 2009). In the program settings, all samples in one qPCR run were treated as one amplicon to set a common window of linearity (r² > 0.999). Subsequently, the program automatically determined the fluorescence threshold for all samples and calculated the individual C_T and efficiencies based on the fluorescence increase, E_fi (Ruijter et al., 2009). The results were exported, and the mean E_fi of each sample was calculated as the arithmetic mean of all replicates. The range of PCR amplification efficiency for individual qPCR reactions using this method ranged from 70 to 104% (E_fi = 1.73 to 2.04). This broad range demonstrates the variability of qPCR efficiency, in other words in amplification quality, with template source (e.g., organic vs. mineral soils). The sub-optimum efficiency reported here may be due to the degenerate primers used to account for the mcrA sequence variability within the methanogen lineage, or the presence of inhibitors that can affect annealing kinetics and the accuracy of the qPCR assay. We used this method to obtain the C_T values for samples based on the linear increase of fluorescence to account for template-related variability of E by correcting for differences in E between the samples and standard (Brankatschk et al., 2012).