References

364 C.C. Tebbe
to obtain PCR-amplifiable material: cell extraction and direct DNA extraction.
Cell extraction means that the bacterial cells are first separated from
thesoilmatrixandcollectedasintactcells.Thesecellsaresubsequently
washedandthencommonprotocolsforextractingDNAfromcultivated
microorganisms can be applied (Holben et al. 1988; Steffan et al. 1988). The
technique requires centrifugation steps and is time-consuming. The yield
of DNA can be relatively low, but the quality, i.e., the purity and size of
DNA fragments of the DNA, is generally high. The alternative approach
is the direct lysis of the microbial cells in the soil matrix followed by the
extraction and purification of DNA (Ogram et al. 1987; Steffan et al. 1988;
Tebbe and Vahjen 1993). The method generates DNA that is initially highly
contaminated with humic material. This DNA must be purified before PCR
can be applied. The key enzyme for PCR, the Taq-polymerase is relatively
sensitive to contamination by humic acids (Tebbe and Vahjen 1993). Direct
extraction of DNA is straightforward and generally well suited for amplifying
DNA for most applications. A large number of different protocols
have been developed to obtain PCR-amplifiable DNA from soil, but to date,
commercially available kits can also be used for this purpose.
The threshold of detecting single genes in soil DNA can be as low as
approx. 10–100 copies/g of soil. In particular, recombinant genes can be
detected efficiently because the specific primers are less likely to crossreact
with DNA of indigenous microorganisms. The most limiting factors
for detecting smaller numbers of genes relate to problems associated with
sample compression and the dimension of a PCR tube. Sample compression
simply means that the total DNA extracted from 1 g of soil, e. g., 80 µg,
can hardly be packed into a single microliter which then would serve as
a template for PCR. In addition, high concentrations of nonspecific DNA
can interfere with the specificity of the primers designed to amplify a low
copy number gene. The dilution of template DNA also alleviates problems
associated with co-extracted compounds such as humic acids. Nevertheless,
for many studies with PCR and soil DNA, it is desirable to use relatively
concentrated DNA instead of dilutions.
5
Cloning, Sequencing and Profiling Marker Genes from Soil
For the detection of more abundant intrinsic marker genes, primer design
becomes a very critical point. Most studies on PCR amplification of
intrinsic marker genes try to retrieve such genes from unknown organisms.
This can be achieved by using primers, i.e., primers which bind to
phylogenetically conserved regions within a selected gene. A bioinformatic
approachbasedonaligningtheknownsequencesofageneisusedtoselect