38 3 Aspects of the Molecular Biology of Microorganisms of Relevance to the Aquatic Environment
the new DNA molecule. For PCR, primers must be
duplicates of nucleotide sequences on either side of the
piece of DNA of interest, which means that the exact
order of the primers’ nucleotides must already be
known. These flanking sequences can be constructed
in the lab, or purchased from commercial suppliers.
The Procedure: There are three major steps in a PCR,
which are repeated for 30 or 40 cycles. This is done on
an automated cycler (thermocycler), which can heat
and cool the tubes with the reaction mixture in a very
short time.
(a) Denaturation at 94°C
The unknown DNA is heated to about 94°C, which
causes the DNA to denature and the paired strands
to separate.
(b) Annealing at 54°C
A large excess of primers relative to the amount of
DNA being amplified is added and the reaction
mixture cooled to allow double-strands to anneal;
because of the large excess of primers, the DNA
single strands will bind more to the primers,
instead of with each other.
(c) Extension at 72°C
This is the ideal working temperature for the
polymerase. Primers that are on positions with no
exact match, get loose again (because of the higher
temperature) and do not give an extension of the
fragment. The bases (complementary to the tem-
plate) are coupled to the primer on the 3¢ side (the
polymerase adds dNTP’s from 5¢ to 3¢, reading the
template from 3¢ to 5¢ side, bases are added com-
plementary to the template). The process of the
amplification in the PCR process is shown in
Fig. 3.4.
3.2.1 Some Applications of PCR
in Environmental Biotechnology
PCR is extremely efficient and simple to perform. It is
useful in biotechnology in the following areas:
(a) To generate large amounts of DNA for genetic
engineering, or for sequencing, once the flanking
sequences of the gene or DNA sequence of interest
is known
(b) To determine with great certainty the identity of an
organism to be used in an environmental biotech-
nology, such as those to be used in bioremediation
(c) To determine rapidly which organism is the cause of
contamination in a production process so as to elim-
inate its cause, provided the primer appropriate
to the contaminant is available3.3 Microarrays
The availability of complete genomes from many
organisms is a major achievement of biology. Aside
from the human genome, the sequencing of the com-
plete genomes of many microorganisms has been com-
pleted and the sequences are now available at the
website of The Institute for Genomic Research (TIGR),
a nonprofit organization located in Rockville, MD with
its website at http://www.tigr.org. At the time of writing,
TIGR had the complete genome of 294 microorgan-
isms on its website (268 bacteria, 23 Archae, and
3 viruses). The major challenge is now to decipher the
biological function and regulation of the sequenced
genes. One technology important in studying func-
tional microbial genomics is the use of DNA
Microarrays (Hinds et al. 2002a).
Microarrays are microscopic arrays of large sets of
DNA sequences that have been attached to a solid sub-
strate using automated equipment. These arrays are
also referred to as microchips, biochips, DNA chips,
and gene chips. It is best to refer to them as microar-
rays so as to avoid confusing with computer chips.
DNA microarrays are small, solid supports onto
which the sequences from thousands of different genes
are immobilized at fixed locations. The supports
themselves are usually glass microscope slides; silicon
chips, or nylon membranes may be used. The DNA is
printed, spotted, or, actually, directly synthesized onto
the support mechanically at fixed locations or
addresses. The spots themselves can be DNA, cDNA,
or oligonucleotides (Fig. 3.5).
The process is based on hybridization probing.
Single-stranded sequences on the microarray are
labeled with a fluorescent tag or flourescein, and hybrid-
ized to, in fixed locations on the support. In microarray
assays, an unknown sample is hybridized to an ordered
array of immobilized DNA molecules of known
sequence to produce a specific hybridization pattern
that can be analyzed and compared to a given standard.
The labeled DNA strand in solution is generally called
the target, while the DNA immobilized on the microar-
ray is the probe, a terminology opposite that is used in