PCR dependent techniques

A denitrifying ability is one of the unique properties of microorganisms. Denitrifying organisms do not belong to a specific taxonomic group and thus total bacterial phylogeny-based approaches are not suitable to study denitrifiers. Therefore, the existing techniques to study the ecology of denitrifier community are based on functional genes (those are responsible for N transformations

during the process) or their transcripts as molecular markers to trace this process (Hallin et al.,

2007; Philippot, 2006; Philippot & Hallin, 2006).

Most culture-independent techniques used to study denitrifiers are based on PCR (Mullis & Faloona, 1987). PCR, comprises cyclic reactions under controlled temperature conditions that lead to the extremely efficient and sensitive amplification of a specific gene region of target DNA (van Elsas & Boersma, 2011). Target DNA is amplified using two primers (reverse and

forward) either universal (e.g. 16S rRNA) or specific (e.g. nirS, nirK, nosZ in this study)

containing sequences complementary to the target region that anneal to opposite ends of the template. DNA polymerase binds to the primer sites and transcribes to the target gene. Repeated temperature cyclings lead to exponential amplification of the target region of DNA. The amplification generates a mixed pool of amplicon when degenerate primers are used or when the target gene is polymorphic, which reflects the composition of the target genes in the studied sample (Philippot & Hallin, 2006).

PCR is subject to various biases such as DNA extraction procedures, primer selection, and PCR conditions. The choice of appropriate DNA extraction method will determine the quality and quantity of the nucleic acid used for further amplification. Inefficient lysis of cells in

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soil samples or improper extraction of DNA will not be representative of the total microbial community present in the sample. Failing to carry out a proper clean up or to remove all the inhibitory compounds will lead to improper or no PCR amplification of the target DNA. Appropriate primer design is crucial for achieving a specific product in order to amplify a wider range to cover as many versions of gene as possible. Appropriate selection of PCR condition, such as annealing temperature, elongation temperature, length of each PCR cycle and number of PCR cycles, is essential for efficient amplification of the target DNA.

Some of the PCR-based community profiling techniques generally used to study denitrifier community structure are described below.

2.5.2.1 Determination of denitrifier community structure

Denaturation/ temperature gradient gel electrophoresis (DGGE and TGGE):

DGGE and TGGE methods were originally developed to detect point mutation in the DNA

sequences; however, these have now been adapted to study microbial ecology (Muyzer et al.,

1993; 1999). TGGE methods are not as popular as DGGE, which have been extensively used to

study denitrifier community diversity in environmental samples (Throbäck et al., 2004). After

PCR amplification, DGGE separates gene fragments of same size on the polyacrylamide gel with a gradient of increasing concentration of the denaturants urea and formamide. Due to its convenience as a rapid, reproducible, and inexpensive method, DGGE allows the analysis of a large number of samples, making it possible to follow changes in the denitrifier communities over time or in response to treatments. DGGE bands can be excised, purified, PCR-amplified, and sequenced so that sequencing of clones can be reduced.

Restriction Fragment Length Polymorphism (RFLP)

This method relies on DNA polymorphism to study denitrifier diversity, also known as amplified ribosomal DNA restriction analysis (ARDRA). In this method PCR-amplified DNA is digested

with 4-base pair cutting restriction enzyme (Liu et al., 1997). The choice of restriction enzyme is

crucial for success of the analysis. After the fragments are cut the fragment lengths are detected using agarose or non-denaturing gradient polyacrylamide gels electrophoresis for community

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communities but not as a measure of diversity or detection of specific phylogenetic groups (Liu

et al., 1997). Since a single species may have 4–6 restriction fragments, it becomes too complex

to analyse species in the banding pattern of diverse communities (Tiedje et al., 1999).

Terminal Restriction Fragment Length Polymorphism (T-RFLP)

This method follows the same principal as RFLP, except for the incorporation of a fluorescently

labelled PCR primer on the 5’ end with a fluorescent dye such as Tetrachlorofluorescein (TET)

or 6-carboxyfluorescein (6 FAM). The mixture of amplified genes, known as amplicons, is then subjected to a restriction reaction, in which an enzyme is used to cut the amplicons at a specific recognition sequence. This sequence results in a mixture of gene fragments that can be separated

based on their length and thus produce a “fingerprint” of the community composition. The

fingerprint can be statistically analysed to produce metrics of the community structure.

Individual bands or peaks are considered “operational taxonomic units” (OTUs), and do not

necessarily correspond to a single species. T-RFLP has been widely used to study denitrifier

community composition in soils (Braker et al., 2001; Rich & Myrold, 2004; Rösch & Bothe,

2005). T-RFLP allows for rapid analysis of the community structure and diversity of functional

genes like nirS, nirK and nosZ in different samples.

2.5.2.1 Determination of denitrifier gene abundance

The abundance of denitrifier genes can be measured using immunological and microarray techniques, but very accurate estimates of gene abundance can be achieved through PCR. In quantitative (or real-time) PCR, gene abundance is measured based on the detection of

fluorescence signals corresponding to the synthesis of PCR amplicons (Heid et al., 1996) allows

for accurate estimation of number of copies of PCR amplicons by extrapolation of the amplicon

accumulation curve (Yoshida et al., 2009). The number of copies of the target DNA is

determined by comparison with a standard curve prepared using DNA of a sample of known

concentration (Yoshida et al., 2009). The two types of commonly used PCR for quantification of

denitrifier genes are competitive PCR (cPCR) and quantitative PCR (qPCR).

The cPCR is based on simultaneous amplification of the target DNA and a known concentration of control DNA known as competitor. The competitor molecule must have the

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same priming sites but a different size from the target gene, making a co-amplification possible

during PCR and detection of target DNA by electrophoresis (Sharma et al., 2007). The

calculation of the amplification of target DNA is based on the ratio of target to competitor PCR

product by agarose gel electrophoresis. cPCR has been successfully used to quantify nirS and

nirK genes in marine, stream sediments and biofilm samples but not been tested for soil (Cole et

al., 2004; Michotey et al., 2000; Qiu et al., 2004).

The key feature of qPCR is that the amplified DNA is detected as the reaction progresses in real time. The quantification at the exponential phase of the PCR when the efficiency is recognised to be the highest is one of the greatest advantages of the qPCR compared with cPCR. Despite numerous alternative probes for qPCR, the most commonly used detectors are non-

specific intercalating dyes (e.g. Syber Green) (Giglio et al., 2003). The dye used in PCR binds

extensively to double stranded DNA (dsDNA) causing fluorescence of the dye. The increase in the formation of dsDNA product during PCR increases the fluorescence of dye and thus the quantification of the DNA content. However, the dye used in PCR may also bind non-specific PCR product including primer-dimers, therefore analysis of the dissociation curve of the samples known as melting curve analysis of the samples must be performed after the PCR amplification

(Sharma et al., 2007). qPCR has been used extensively to study the influence of soil

environmental factors such as O2 status, pH, temperature, nutrient availability on denitrifier gene

abundance (López-Gutiérrez et al., 2004; Miller et al., 2008; Philippot, 2005; 2006; Philippot &

Hallin, 2006; Philippot et al., 2009).