in Bacteria and Archaea or 18S in Eucarya; the large subunit (LSU) rRNA genes, 23S in Bacteria and Archaea or 28S in Eucarya; or the internal transcribed spacer (ITS) regions, sequences that lie between the SSU and the LSU genes. Other defined targets are genes that code for ecologically significant functions, such as genes that code for proteins involved in nitrogen fixation, e.g., nif H, which encodes the iron protein of nitrogenase reductase; amo A, which codes for ammonium monooxygenase, a key enzyme in nitrification reactions; and nirS, which codes for nitrite reductase, a key enzyme in denitrification reactions.
In any study in which PCR is used, sources of bias must be considered (v. Wintzingerode et at., 1997). The main sources of bias in amplifying soil community DNA are: (i) the use of very small sample sizes (typically only 500 mg of soil), which may represent only a small fraction of the whole soil community; (ii) preferential amplification of some DNA templates over others due to the greater ease of binding of DNA polymerase to some sequences over others; and (iii) for amplification of the rRNA genes, the fact that many bacteria contain multiple copies of these operons (e.g., Bacittus and Ctostridium species contain 15 copies), hence sequences from such species will be overrepresented among the amplification products. In addition, chimeras, composed of double-stranded DNA in which each strand was derived from a different organism rather than a single organism, may be generated. This latter problem is sometimes a consequence of using too many cycles in the PCR. Acknowledged biases associated with PCR are generally why diversity indices calculated from the results of PCR-based experiments may not be very robust and their use is not encouraged.
An advance in PCR analysis that allows specific gene targets to be quantified is quantitative PCR (qPCR), also called real-time PCR. qPCR is a method that employs fluorogenic probes or dyes to quantify the number of copies of a target DNA sequence in a sample. This approach has been used successfully to quantify target genes that reflect the capacity of soil bacteria to perform given functions. Examples include the use of ammonia monooxygenase (amoA), nitrite reductase (nirS or nirK), and particulate methane monooxygenase (pmoA) genes to quantify ammonia-oxidizing (Hermansson and Lindgren, 2001), denitrifying (Henry et al., 2004), and methanotrophic (Kolb et al., 2003) bacteria, respectively, in soil samples. qPCR coupled with primers to specific ITS or rRNA gene sequences has also been used to quantify ectomycorrhizal (Landeweert et al., 2003) and endomycorrhizal fungi (Filion et al., 2003) as well as cyst nematodes (Madani et al., 2005) in soil.
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