Environmental Engineering Reference
In-Depth Information
3. Complementary strand synthesis by primer extension of each of the single
strands produced by step 1 at a temperature of 75
◦
-80
◦
C
The three-step procedure is repeated usually 30 to 40 times in order to obtain
exponential copies of PCR product. The two important variables in successful
use of PCR as a tool are primer synthesis or selection and PCR operating con-
ditions. These two factors dictate the level of specificity and sensitivity that can
be obtained by PCR and are instrumental in facilitating the detection of target
nucleic acids at refined taxonomic levels.
Evaluation of PCR Products
The purpose of amplifying target nucleic acids
present in the environmental sample is to be able to subject a sufficient quan-
tity of the representative material (PCR product) to a laboratory procedure for
the determination of the microbial agent that it represents. Classic procedures
for this purpose involve application of a series of concentrations of the PCR
products to an agarose gel electrophoresis slab along with a molecular marker.
Various amplified gene fragments migrate through the gels in proportion to their
molecular weights. The separated gene fragments can then be confronted with an
oligonucleotide probe specific for the organism of interest in relation to its possi-
ble presence in the original water sample. Oligonucleotide probes are conjugated
with a reporter molecule (typically a fluorogenic compound) that under appropri-
ate conditions (fluorescent lighting) signals hybridization with a complementary
(target) nucleic acid fragment.
Two areas of interest in connection with molecular detection of specific micro-
bial agents in environmental samples are robustness of the detection effort and the
level or density of the target microbe in the representative environmental sample.
In the former, since molecular detection is a gene-based exercise, it stands to
reason that the more types of gene fragments that are available as probes, the
more information that can be learned about the genome of the target organism.
The technique that makes use of the multiple probe approach is the microarray.
The microarray is a glass microscope slide that serves as a solid support for
the spotting of literally thousands of genes or gene fragments — in this example,
oligonucleotide probes — that serve to test hybridization potential with amplified
gene fragments (PCR products) of unknown identity. The nucleotide sequence of
the probe is known and representative of specific microbes. The location of each
of the probes on the glass slide is carefully recorded, so when hybridization with
unknown PCR products (amplicons) is indicated by reporter signals, the strain,
species, and genus identity of the unknown amplicon can be learned.
Quantification of the target microbe in the environment with the aid of a PCR
instrument must involve procedural modifications and special equipment in order
to measure the level of production of PCR products. Fluorogenic probes and a
fluorescence detection device are used to track the formation of PCR product for-
mation. Quantitative PCR (qPCR) is still relatively new, and advances are being
made to increase its utility. The following brief description is based on methodol-
ogy described by Grove.
142
In the qPCR process, two fluorogenic probes anneal
