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Kathryn L. Dudeck and Wendy A. Dustman
University of Georgia,
Department of Marine Sciences
Introduction
This application describes the use of the MasterTaq polymerase kit for
improving PCR efficiency when studying functional gene presence. Using
this polymerase, it was possible to amplify a 675 bp region of the amoA
gene in lab-grown Nitrosococcus oceani, which has previously been met
with many obstacles when utilizing other commercially available enzyme
kits.
The Polymerase Chain Reaction (PCR) is a now-commonplace method used for
making several copies of a specific gene for further manipulation, or
for simply observing whether or not a specific gene is present.
Lab cultures of Nitrosococcus oceani and other ammonia-oxidizing bacteria
(AOB) are notoriously difficult to study. They exhibit slow growth and
therefore are easily contaminated by hetero t rophs. In addition to this
hurdle, the gene used in this study is often found with three clustered
subunits (amoA, amoB, and amoC) within some members of AOB (Alzerecca
et. al, Sayavedra-Soto et. al). The gene lengths are remarkably similar;
there f o re, the study of a single gene in
this area of the chromosome can be quite challenging.
Materials and Methods
Bacterial Preparation
Nitrosococcus oceani (ATCC 19707) was cultured in ATCC Medium 928 in a
shaking incubator (100 rpm) at 27 C. Using 12.5% K2CO3, the pH level
was maintained at ~8.3. Twenty-five milliliters were extracted and purified
using a Qiagen blood Mini Prep Kit.
PCR Amplification
Two bacterial primers, A189, GGN GAC TGG GAC TCC TGG (Holmes et. al) and
AmoA2-R, CCC CTC KGS AAA GCC TTC TTC (Rotthauwe et. al), were utilized
to amplify a 675 bp region of the amoA gene of the cultures. PCR was performed
as follows: 2 l of each primer (10 M), 1 l of N.
oceani culture, 2.5 l of 10X buffer, 2 l of 2.5 mM dNTP,
2.5 U of Taq polymerase, and 0-5 l of 5X TaqMaster. This mixture
was adjusted to a final volume of 25 l using
sterile water. A negative control (no DNA) was also amplified.
Amplification of the target gene was carried out via the following program:
94 C for 2 minutes, 35 cycles of 94 C for 1 minute, 56 C
for 1 minute, and 72 C for 1 minute, and a final extension step of
56 C for 5 minutes.
Visualization
PCR products were run in a 2.0% agarose gel and stained with ethidium
bromide for visualization.
Results and Discussion
The 675 bp region of the amoA gene was successfully amplified using the
Eppendorf MasterTaq polymerase kit. Varying the amount of TaqMaster
thermostabilizer included in the PCR reaction had a minimal effect on
the band obtained from N. oceani. The addition of 2.5 l of TaqMaster
gave the most favorable results (Lane 4), though bands were visible at
all concentrations of TaqMaster (Lanes 3 and 5). The additional bands
seen in the N. oceani lanes are most likely the amo subunits. Internal
probing after PCR verifies that the band seen at approximately 675 bp
is indeed amoA (data not shown). Eppendorfs MasterTaq polymerase
kit is a beneficial enzyme system to use when attempting to study known
laboratory cultures of ammonia-oxidizing bacteria.
Lane 1 - Molecular weight marker VIII
Lane 2 - Negative control (no DNA)
Lane 3 - Nitrosococcus oceani with no Ta q M a s t e r
Lane 4 - Nitrosococcus oceani with 2.5 l Ta q M a s t e r
Lane 5 - Nitrosococcus oceani with 5 l Ta q M a s t e r
References
Alzerecca, J.J., J.M. Norton, and M.G. Klotz. 1999. The amo operon in
marine, ammonia-oxidizing gamma-proteobacteria. FEMS Microbiology Letters
180:21-29.
Holmes, A. J., A. Costello, M. E. Lidstrom, and J. C. Murrell. 1995. Evidence
that particulate methane monooxygenase and ammonia monooxygenase may be
evolutionarily related. FEMS Microbiology Letters. 132:203-208.
Rotthauwe, J. H., K. P. Witzel, and W. Liesack. 1997. The ammonia monooxygenase
structural gene amoA as a functional marker: molecular fine-scale analysis
of natural ammonia-oxidizing populations. Applied and Environmental Microbiology.
63:4704-4712.
Sayavedra-Soto, L.A., N.G. Hommes, J.J. Alzerreca, D.J. Arp, J.M. Norton
and M.G. Klotz. 1998. Transcription of the amoC, amoA and amoB genes in
Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiology Letters
167:81-88.
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