In order to determine the abundance of gene copies or transcripts in an environmental sample real-time PCR is a commonly used method as it enables the specific detection of PCR products in real-time during the exponential amplification stage of the reaction. This method is based on the detection of fluorescence signals and distinguishes between probe-based chemistries (TaqMan®, Molecular Beacons, Scorpions®, FRET Probes) and the SYBR® green chemistry (Fig. 6).
Figure 6. SYBR® green chemistry: Reporter dye (R) starts fluorescing as soon as it intercalates double-stranded products.
When SYBR® green (Publication II, III) is added to a reaction, it intercalates all double-stranded DNA present in the sample and binds to each new copy of double- stranded DNA generated during the amplification process (Fig. 6). To achieve an absolute quantification, standard curves have to be created by amplifying known amounts of DNA in a parallel group of reactions run under identical conditions to that of the sample. Additionally, a reference dye, called ROX, serving as internal standard, is used in each sample to normalize the well-to-well difference arising through artefacts like pipetting errors and instrument limitations. The primary disadvantage of the SYBR® green assay is that unspecific products may be detected as well as SYBR® green intercalates any double-stranded product (Wittwer
et al.,
However, the performance of a dissociation curve analysis, generating melting peaks of the amplicons, allows the distinct differentiation between the targeted product and undesired products like contaminations and primer dimers (Fig. 7) (Rasmussen et al.,
1998). Furthermore, in case of environmental samples, divergences in the GC content of a specific gene, present in different organisms, may lead to the formation of multiple peaks (Fig. 7). Specificity of the reaction must then be confirmed on an agarose gel (Fig. 8).
Figure 7. Melting profiles of a typical PCR run (in this case nirK gene quantification; Publication II)
showing a) specific products of the standard, b) multiple peaks of the samples due to divergences in the GC content in the different organisms of a sample and c) the negative controls, giving no melting peaks. Furthermore, indicated is the melting profile of a primer dimer (d) which may occur between 70°C and 75°C.
Figure 8. Agarose gels of the amplified nirK genes from Fig. 7, showing the specificity of the
reaction with fragments of the targeted length (164 bp): standards in position 1-5, samples in position 6-23 and the negative control in position 24.
a)
b) d)
few. All these factors have the potential to influence the specificity of the PCR reaction. Low efficiencies increase the number of needed cycles and may lead to the amplification of unspecific products inhibiting the amplification of the targeted product due to competition. Therefore, the selection of an appropriate primer system is of high importance. Ideal primer sets generate amplicons of less than 150 bp
(Tichopad et al., 2002). Using the PCR efficiency as quality criterion
(E = (10 (-1/slope) – 1) * 100%; (Pfaffl, 2001)), short fragments may exhibit values between 90% – 110%. However, primer systems targeting functional genes from environmental samples usually produce amplicons of 150 bp – 500 bp yielding PCR efficiencies between 80% – 120% (-3.9 > slope > -3.0). Due to the mentioned requirements for amplification products, most of the existing primer systems cannot be used; only a few primer sets are verified and approved for the quantification of functional genes from environmental samples, which have been used in this thesis (Publication II and III).
Another consideration includes the possible existence of multiple gene copies per cell, which affects the actual community abundance and thus aggravates the interpretation of qPCR results. Experimental data indicate multiple nifH gene copies
in Paenibacillus azotofixans (Rosado et al., 1998) and in Anabaena oscillarioides
(Kirshtein et al., 1991). But due to little information on genome copy variability per
cell in different microorganisms, one nifH copy per microbial genome was assumed
in this thesis (Publication II). Likewise, little is known about the genome structure of archaeal ammonia oxidizers. Based on existing data for AOA (Könneke et al.,
2005; Treusch et al., 2005), one amoA copy per organism was assumed
(Publications II and III). Research on AOB revealed two amoA copies in Nitrosomonas spp. and three copies for Nitrosospira spp. (Norton et al., 2002), for
which reason 2.5 amoA copies per AOB were calculated in this thesis (Publication
II). Concerning the denitrifier population, gene copy numbers can be directly correlated with the community size, as no organism has been found yet to possess multiple copies of corresponding nitrogen cycle genes, except nitrate reducers which may harbour two narG copies per organism (Philippot et al., 2002). The nirS and nirK copy numbers were therefore directly referred to the number of nitrite reducers
The quantification of functional microbial communities in the environment by real- time PCR has been applied in many studies, which proves the potential for quantitative applications in the environment: e.g. quantification of nitrifiers in terrestrial (Adair & Schwartz, 2008; Boyle-Yarwood et al., 2008) and aquatic
ecosystems (Wuchter et al., 2006), quantification of denitrifiers (Henry et al., 2008;
Kandeler et al., 2006) and nitrogen fixers (Coelho et al., 2009) in soil. In
Publication II, key genes involved in nitrogen cycling (nifH, AOB-amoA, AOA- amoA, nirS and nirK) were detected using the SYBR® green assay reflecting the
nitrogen-fixing, ammonia-oxidizing and denitrifying communities. In Publication III, key genes of the nitrification pathway (AOB-amoA, AOA-amoA) have been
quantified and referred to community structure and activity in order to assess the adaptation to a new management system.