MICROBIAL MONITORING DURING BIOAUGMENTATION WITH DEHALOCOCCOIDES - Bioaugmentation for Groundwater Remediation - page 178

generally as DNA polymerases and they function during replication of microbial DNA.
Replication using DNA polymerases occurs rapidly and reasonably accurately, with error rates
less than 1 in 10,000 basepairs. In addition to the DNA polymerase, PCR requires small fragments
of DNA called oligonucleotide primers (or simply just “primers”) in order to target specific DNA
sections of interest within a microbial genome. These primers are complementary to short
sections of DNA, are usually 20-30 basepairs in length, and used in pairs to amplify genes of
interest. These genes range from species-specific indicators, such as 16S ribosomal RNA (rRNA)
genes (found in all microbes), to functional genes such as those responsible for specific metabolic
reactions like ammonia-oxidation (e.g., the amoA gene) or VC reduction (e.g., vcrA ).
Some genes, like the 16S rRNA, contain highly conserved portions of DNA (i.e., common
among a large group of different microbes) interspersed with highly variable sections of DNA.
The conserved regions allow the design of primers to amplify the DNA, while the variable
regions permit molecular analyses useful in identifying closely related microbes. Genes such as
vcrA , which codes for the catalytic subunit of the enzyme responsible for the reductive
dechlorination of vinyl chloride, are less conserved, but are found in only a limited number
of microorganisms. As described below, this conserved and variable nature of various DNA
sections may affect data interpretation, especially when analyzing environmental samples.
PCR consists of a number of cycles (commonly 25-50), each composed of three steps, with
each step occurring at a different temperature. A device named a thermocycler is used to
automatically adjust the temperatures required for each step. The three steps are: (1) an initial
denaturing step, (2) an annealing step, and (3) an extension step. The denaturing step uses
elevated temperatures to separate double-stranded DNA into two single-stranded pieces. Once
the DNA is denatured, the temperature is rapidly decreased and the two primers anneal along
complementary matches on the single-stranded DNA. The primers guide the DNA polymerase
towards the targeted sequence, permitting the beginning of DNA replication, which occurs in
the extension step. During the extension step, the temperature is increased and the DNA
polymerase adds complementary nucleotides, which are added as part of the reaction mixture,
based upon the sequence of the original DNA strand. These newly synthesized fragments serve
as the DNA templates for the next round of reactions. At the end of each three-step cycle, the
number of synthesized DNA fragments is double the previous quantity, producing extremely
large working concentrations of DNA from relatively small starting concentrations. Thus, PCR
is an extremely sensitive technique for detecting small numbers of genes.
qPCR functions in a manner similar to PCR, but through the use of fluorescent probes,
qPCR allows quantification of the amplified DNA sequence. A qPCR probe is similar to the
primers described above, except that it contains a fluorescent reporter at one end and a
quencher at the opposite end. As with the primers, this probe binds along complementary
DNA during the annealing step. When the fluorescent reporter and the quencher are in close
proximity, such as at the probe ends, no signal is emitted. However, as the DNA polymerase
adds nucleotides and moves along the DNA strand during replication, the fluorescent reporter
and quencher are cleaved from the probe and separate. The reporter subsequently emits a
fluorescent signal, which is detected and quantified in the thermocycler at the end of every
cycle. A larger fluorescent signal corresponds to a greater number of cleaved fluorescent
reporters. Since one probe containing a single fluorescent reporter hybridizes with a single copy
of DNA, the resulting signal strength after the reporter releases from the probe is directly
proportional to the number of DNA copies present at the end of a cycle. A comparison of the
fluorescent signal strength against standard curves permits the quantification of specific DNA
sequences within complex samples.
Over the course of the qPCR cycles, the fluorescent signal strength typically appears
sigmoidal, with an exponential increase, followed by a linear increase, an exponential decrease
and finally a plateau. A point is selected from this curve where the fluorescent signal strength is