References

Marker Genes in Soil Microbiology 365
such primers. Most knowledge in this regard has been collected from work
with SSU rRNA genes, and a large number of primers with specificities for
different phylogenetic lineages, groups or clusters is now available. A PCR
reaction with primers hybridizing to evolutionary conserved regions of
the SSU rRNA genes and soil DNA normally yields PCR products having
the same size, but different nucleotide sequences (due to the variable and
hypervariable regions). A general strategy to sequence different SSU rRNA
genes from soil is to clone the PCR products in E. coli, screen the cloned
products for differences, e.g., with restriction endonucleases, and sequence
the different products.
The first primers allowing the amplification of bacterial SSU rRNA genes
from noncultivated bacteria were described in 1990 (Edwards et al. 1990;
Giovannoni et al. 1990) and their first applications on environmental DNA
already indicated that the majority of sequences were derived from unknown
or not-yet cultured microorganisms. The first SSU rRNA genes that
were amplified from soil DNA were reported by Liesack and Stackebrandt
in 1992 (Liesack and Stackebrandt 1992). Since then, many publications
havereportedonthediversityofSSUrRNAgenesfromsoilandstill,even
with public databases containing more than 35,000 rRNA gene sequences,
the majority of sequences that are isolated from soil are typically not identical
to these sequences. In addition, the closest relatives that can be found
for newly recovered sequences are often SSU rRNA genes of other uncultured
microorganisms. We know today that many soil bacteria and archaea
belong to phylogenetic groups with only a few cultivated organisms, e. g.,
Acidobacteria, Verrucomicrobia or Planctomycetes. Other sequences that
are frequently recovered from soil samples fall into phylogenetic groups
like TM7, WS6, or OP11, which are to date exclusively represented by sequences
of noncultivated organisms (Borneman et al. 1996; Bintrim et al.
1997; Dojka et al. 1998; Hugenholtz et al. 1998). Indeed, one of the major
objectives for soil microbiological research today is to succeed in cultivating
representative organisms from these groups or to study the structure
of their genomes without cultivation.
The cloning and sequencing of PCR products amplified from soil DNA
are excellent strategies for assessing the total microbial diversity in a soil
sample. However, for many ecological studies, different samples need to
be compared with each other, e.g., to characterize an effect of a certain
treatment or environmental change. In such situations, genetic profiling
techniques are very useful, as they generate genetic fingerprints from the
diversity of PCR products amplified with universal primers. Genetic profiling
techniques allow the inclusion of larger numbers of replicates, which
is important in differentiating actual effects from the natural variability
of a microbial community. The most commonly applied techniques
for generating genetic profiles are DGGE (denaturing gradient gel elec-