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Applied and Environmental Microbiology, November 1998, p. 4522-4529, Vol. 64, No. 11
College of Oceanic and Atmospheric
Sciences1 and
Department of
Microbiology,2 Oregon State University,
Corvallis, Oregon 97331
Received 26 March 1998/Accepted 9 September 1998
Marine bacterioplankton diversity was examined by quantifying
natural length variation in the 5' domain of small-subunit (SSU) rRNA
genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast.
This new technique, length heterogeneity analysis by PCR (LH-PCR),
determines the relative proportions of amplicons originating from
different organisms by measuring the fluorescence emission of a labeled
primer used in the amplification reaction. Relationships between the
sizes of amplicons and gene phylogeny were predicted by an analysis of
366 SSU rDNA sequences from cultivated marine bacteria and from
bacterial genes cloned directly from environmental samples. LH-PCR was
used to compare the distribution of bacterioplankton SSU rDNAs from a
coastal water sample with that of an SSU rDNA clone library prepared
from the same sample and also to examine the distribution of genes in
the PCR products from which the clone library was prepared. The
analysis revealed that the relative frequencies of genes amplified from
natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product
molecules can skew gene frequencies when PCR product concentrations exceed threshold values.
Libraries of small-subunit rRNA gene
(SSU rDNA) clones prepared by PCR are widely applied to study the
microbial diversity of natural ecosystems. These studies have provided
dramatic evidence that the majority of microbial communities are
dominated by previously unknown organisms (4, 8, 18).
However, quantitative comparisons using clone libraries to assess
microbial community structure have been limited by several factors,
including (i) undersampling of diversity and (ii) uncertainty about
sources of bias in the cloning process, in particular bias by the PCR.
Undersampling, often estimated by coverage values or by rarefaction
curves, results from the difficulty of processing a large number of
clones (11). The lack of an alternative means to
quantitatively assess the composition of complex mixtures of rDNAs from
in situ communities has made it difficult to evaluate methodological
sources of bias by the cloning process. The method we describe here,
length heterogeneity analysis by PCR (LH-PCR), overcomes some of these
problems by quickly providing a profile of amplicon diversity in
complex mixtures of PCR products.
LH-PCR is similar to the approach used in our earlier study of bias in
the PCR (16) and the recently published terminal restriction
fragment length polymorphism (9) and fluorescent restriction
fragment length polymorphism (2) techniques. In both LH-PCR
and these methods, the proportions of PCR amplicons originating from
different genes are estimated from the fluorescence emission of labeled
PCR primers. However, instead of identifying PCR amplicons based on
restriction endonuclease sites, in LH-PCR the discrimination of
amplicons originating from different organisms is based on natural
variation in the lengths of SSU rDNAs.
In a previous study, we investigated biases introduced during the
amplification of rDNAs by PCR (16). In that study, the templates consisted of pairwise mixtures of SSU rDNAs from bacteria belonging to three different phylogenetic groups. To estimate bias, we compared the proportions of genes in the PCR products with their proportions in the starting template mixtures. We observed that, above threshold product concentrations, PCR dramatically biased
the frequency distribution among gene homologs relative to the original
mixture. A kinetic model based on competition between primers and
products which successfully explained the experimental results was
developed. These results indicated that this type of bias by PCR might
lead to an increase in net diversity estimates among amplicons relative
to the gene diversity of the native DNA mixture. Evidence also
indicated that artifacts resulting from this phenomenon could be
controlled by limiting the number of replication cycles to maintain
product levels below threshold values. However, the effect of this type
of bias on the composition of PCR products amplified from natural
community DNA was uncertain since, in a complicated mixture of genes, a
single gene might not reach threshold concentrations at which
competition between product and primer reannealing would have a
pronounced effect.
Here we present the results of a study in which LH-PCR was used to
estimate the community composition of bacterioplankton from a water
sample collected off the Oregon coast. In order to trace the
phylogenetic origin of the domains amplified by LH-PCR, we performed an
analysis of the length variability in SSU rDNAs of bacterial strains
cultivated or directly cloned from the same seawater sample, as well as
sequences retrieved from gene sequence databases. We found that
the relative gene frequencies obtained from natural communities
by LH-PCR were highly reproducible when PCR product concentrations were
limited to relatively low values but that, at high concentrations, the
kinetic bias caused by template reannealing significantly skewed gene
frequencies. SSU rDNA amplicons with sizes corresponding to the alpha
subdivision of the class Proteobacteria
(alpha-Proteobacteria) represented the largest fraction of the bacterial rDNA amplicons (ca. 65%), while no other size class of SSU rDNA amplicons represented greater than 10% of
the bacterial rDNA amplicons. Overall, the results suggest that
LH-PCR is an effective tool for assessing microbial community structure and that clone libraries may often overrepresent
bacterioplankton diversity because the relative frequencies of dominant
species have been reduced by a systematic bias.
Sample collection, nucleic acid isolation, and clone library
construction.
On 28 April 1993, a subsurface (10-m) water sample
was collected by Niskin bottles at a station located 8 km off the mouth of Yaquina Bay, Oreg. (44°39.1'N, 124°10.6'W). The water was
prescreened through 10-µm-pore-size Nitex mesh and transported in
autoclaved polyethylene carboys to the laboratory for the remaining
analyses. Picoplankton from 4-liter (subsample 1) and 16-liter
(subsample 2) subsamples were collected by filtration onto
0.2-µm-pore-size polysulfone filters (Supor-200; Gelman Sciences
Inc., Ann Arbor, Mich.). Total cellular nucleic acids were
isolated from the picoplankton samples by lysis with proteinase K and
sodium dodecyl sulfate, followed by phenol-chloroform extraction as
previously described (6). A portion of the DNA sample
isolated from subsample 2 was used as template in the amplification of
nearly full-length SSU rDNAs by PCR and subsequently cloned into a
plasmid vector as described elsewhere (12, 17). SSU rDNA
clones recovered in this library have been partially described
elsewhere (12, 17).
LH-PCR.
Ten nanograms of purified genomic DNA from each
subsample was used as template for LH-PCR. The forward primer, 27F
(5'-AGA GTT TGA TCM TGG CTC AG-3') (3), was 5' end labeled
with the phosphoramidite dye 6-FAM (graciously supplied by Applied
Biosystems Inc., Foster City, Calif.) or purchased from Genset (San
Diego, Calif.). The reverse primers used were 355R (5'-GCT GCC TCC CGT AGG AGT-3') (1) for domain A and 536R (5'-GWA TTA CCG CGG
CKG CTG-3') (5) for domain B, synthesized at the Central
Services Laboratory, Center for Gene Research and Biotechnology, Oregon State University. In a final volume of 100 µl, reaction mixtures contained 0.2 mM premixed deoxynucleoside triphosphates (Stratagene, La
Jolla, Calif.), 1.5 mM MgCl2, 5% acetamide, 0.5 µM
forward primer, 0.5 µM (one) reverse primer, and 2.5 U of
Taq DNA polymerase (Promega, Madison, Wis.). All reactions
used the Ampliwax hot-start protocol (Perkin-Elmer Cetus, Norwalk,
Conn.) in a PTC100 thermal cycler (MJ Research Inc., Watertown, Mass.)
programmed to 16 cycles for primer 355R (except for the reactions
evaluating PCR bias) or 21 cycles for primer 536R, each consisting of
96°C denaturation for 1 min, 55°C annealing for 1 min, and 72°C
extension for 3 min.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Kinetic Bias in Estimates of Coastal Picoplankton Community
Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR
Amplicon Length Heterogeneity
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Length heterogeneity analysis of published sequences. In LH-PCR, amplicons originating from different templates are identified by length heterogeneity in hypervariable regions of the SSU rDNA. Three such regions occur in the 5' end of the gene, around locations homologous to Escherichia coli positions 90, 190, and 450. In order to verify the phylogenetic coherence of length heterogeneity contained in these variable regions, we compared the length heterogeneity of domains homologous to the domain between E. coli positions 8 and 355 (domain A) and positions 8 and 536 (domain B). The analysis included previously published sequences of bacterial species isolated from the same water sample as that used for the LH-PCR analysis or directly cloned from DNA extracted from subsample 2 (17), as well as SSU rDNA sequences of bacterial species isolated from seawater or directly cloned from DNA extracted from seawater, retrieved from the GenBank, Ribosomal Database Project (10), and ARB (14) sequence databases.
Bias by PCR. Two experiments were performed to evaluate the introduction of bias by PCR. In order to evaluate the bias described by Suzuki and Giovannoni (16) in the amplification of domain A (16) and to optimize the number of cycles for LH-PCR, we performed a time course experiment in which PCRs of domain A, with DNA purified from subsamples 1 and 2 as templates, were stopped by freezing at 10 (only subsample 1), 12, 14, 16, 18, 20, and 25 cycles. Concentrations of LH-PCR products from subsample 1 were measured spectrophotometrically as described above. Concentrations of LH-PCR products from subsample 2 were estimated from the agarose minigel as described above, except for the products of reactions with subsample 2, and stopped after 12 cycles, which were calculated assuming an amplification efficiency of 85% per cycle (13).
To evaluate the introduction of reannealing bias by PCR in the amplification of full-length rDNAs from mixed populations of bacteria, we used the optimized LH-PCR protocol for domain A as described above to compare the genotypic bacterioplankton community structure of (i) a genomic DNA sample from the Oregon coast (subsample 2) and (ii) the nearly full-length PCR products from subsample 2 after 35 cycles of amplification, used to prepare the SSU rDNA clone library, as described previously (12, 17). Triplicate LH-PCRs were performed as described above with 10 ng of genomic DNA or 60 pg of nearly full-length SSU rDNA PCR amplicons as templates, calculated so that the reactions using genomic DNA and full-length PCR amplicons contained approximately the same numbers of copies of SSU rDNAs. For this calculation, we assumed a bacterial origin for 50% of the DNA, an average chromosome size of 2 Mbp, and an average of two copies of the ribosomal operon per chromosome. Finally, to estimate the introduction of bias by the cloning per se, we compared the community structure estimated by LH-PCR from full-length PCR products amplified from subsample 2 to that inferred from the relative proportion of SSU rDNA clones recovered in the clone library, grouped according to the sizes of domain A obtained directly from their SSU rDNA sequences.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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Predicted length heterogeneity in the 5' region of SSU rDNAs. Three regions at the 5' end of the SSU rDNA (V1, E. coli SSU rDNA positions 72 to 101; V2, E. coli SSU rDNA positions 176 to 221; and V3, E. coli SSU rDNA positions 451 to 481) are variable between different phylogenetic groups of bacteria. Insertions and deletions in these variable regions cause natural variability in the nucleotide lengths of molecules amplified with the 27F and 355R primer pair (domain A, ca. 312 to 363 bp) and the 27F and 536R primer pair (domain B, ca. 472 to 574 bp).
The lengths of domains A and B of bacteria isolated from seawater or SSU rDNAs directly cloned from seawater DNA are shown in Table 1 and are generally coherent with phylogenetic relationships. Many discrete fragment lengths are monophyletic but are shared by multiple species (e.g., 316 bp). Alpha-Proteobacteria and cyanobacteria have the shortest lengths for both domains. Beta-, gamma-, and delta-Proteobacteria and the Flexibacter-Bacteroides-Cytophaga group have intermediate lengths, and the longest domains are those from genes of low- and high-G+C gram-positive bacteria and members of the Vibrio fischeri subgroup of the gamma-Proteobacteria. Most phylogenetic groups have a unique combination of lengths for domains A and B (i.e., alpha-Proteobacteria have a domain A length of 315 bp and domain B lengths between 470 and 472 bp). The lengths of domains A and B of genes with plastid origins were not included in this study and are described elsewhere (12).
|
Analyses of coastal bacterioplankton diversity. An example of an LH-PCR electropherogram is shown in Fig. 1. It shows the length heterogeneity of domain A for PCR products obtained directly from DNA extracted from seawater subsample 2. The 23 peaks are labeled A through W and correspond to amplicons with varying lengths in domain A. Organisms that produce amplicons corresponding in size to these peaks were identified by reference to a clone library prepared from the same seawater sample. These clones are indicated in Table 1 by the prefix "env.OCS."
|
|
Bias by PCR. The possibility that a kinetic bias caused by template reannealing could occur during the amplification of domain A from bacterioplankton samples was investigated by examining the relationship between the final concentration of products obtained and the relative frequency of dominant genes in the population. A portion of this analysis is provided in Fig. 3, which shows the relative frequency of the 317-bp fragment (alpha-Proteobacteria and prymnesiophyte plastids) as a function of the total product molarity. The prediction for the kinetic bias effect is that the proportion of the dominant peak (the percentage of integrated fluorescence) should decrease with increasing product molarity, as observed in Fig. 3. This prediction assumes that the dominant peaks are composed primarily of genes of one or a few types. The final concentrations of product amplicons for the reactions used for Fig. 3 varied for the two samples of DNA isolated independently from the same water sample (subsamples 1 and 2) and also varied according to the number of cycles used for the amplification (12 to 25 cycles for subsample 1 and 12 to 18 cycles for subsample 2). We considered the possibility that biases caused by primer selection, which are dependent on the number of cycles, might have caused the bias. A plot of the same data shown in Fig. 3 with the number of amplification cycles replacing the final product concentrations on the abscissa revealed no relationship (data not shown). The observed bias is in accord with predictions for the kinetic bias effect.
|
Comparison of domain A with domain B. In general, there was good agreement between the community structure estimated by LH-PCR for domains A and B (data not shown). The main difference between LH-PCR for domains A and B was the resolution of different peaks by the Genescan software. Genescan resolved more peaks in the analysis of domain A and tended to merge domain B peaks, especially peaks for larger fragments (peaks > 520 bp). Some adjacent peaks of domain A were also merged in some of the electropherograms (peaks C and D, E and F, K and L, and Q and R).
Phylogenetic composition of the community. The relative proportions of LH-PCR peaks conform to previous observations that alpha-Proteobacteria dominate SSU rDNA clone libraries of surface samples. Peaks A to E and K to M, which correspond to alpha-Proteobacteria and plastids, respectively (Table 1), represent about 65% of the total fluorescence. Peaks F, G, and L correspond to plastid sequences (12). Most of the remaining peaks do not correspond to coherent phylogenetic groups when reference is made to all SSU rDNA sequences of bacteria isolated or environmental clones from seawater. However, most of the peaks had a corresponding isolate or environmental SSU rDNA clone from the same seawater sample. Peaks P and Q, which represented about 7% of the total fluorescence, corresponded to the sizes of beta-, gamma-, and delta-Proteobacteria, Flexibacter-Bacteroides-Cytophaga, and high-G+C gram-positive bacteria, many of which are cultivated strains. Peak S represented about 5% of the total fluorescence and corresponded to the sizes of previously cultivated members of the gamma-Proteobacteria. Finally, peaks T to V represented about 9% of the total fluorescence and corresponded to the sizes of several phylogenetic groups.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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The adoption of molecular techniques for assessing microbial diversity has engendered a far-reaching appreciation of the importance of uncultured microbes but has also led to concerns about the limitations of the new methodologies. The approach we employed here, LH-PCR, was designed to address some of these concerns in the context of the study of complex natural communities.
We found that gene frequencies measured by PCR amplification can be
highly reproducible. We also observed a bias that selectively reduced
the relative frequency of a dominant size class of fragments as a
function of increasing final product concentrations, which can be
explained by the template reannealing bias described by Suzuki
and Giovannoni (16). This bias is caused by the fact that,
as the reaction progresses, amplicons increase in concentration and primers decrease in concentration. At a certain point, amplicons should reanneal, inhibiting the primers from annealing and stalling the
reaction. Assuming that different SSU rDNAs do not cross-reanneal, genes with higher initial concentrations in the original sample should
experience template reannealing at lower combined product concentrations
which for reactions run at the same initial conditions should be dependent on the number of replication cyclesthan genes with lower initial concentrations. Therefore, in reactions experiencing this bias, dominant species should be underrepresented and rarer species should be overrepresented. The fact that the observed trend was
related to combined product concentration but not to the number of
amplification cycles supports the idea that this bias is due to
reannealing kinetics and not to some other form of bias, such as primer
selection. This reannealing bias became significant above product
concentrations of about 2 nM, and therefore, we recommend that
amplification reactions be stopped before reaching this value.
Differences between the LH-PCR electropherograms obtained from natural community DNA and those obtained from the reamplification of full-length SSU rDNA amplicons indicate that the PCR may significantly bias the composition of clone libraries. However, such biases do not appear to occur randomly but rather are systematic. The shift in the dominance from the peak of 317 bp to the peak of 319 bp contradicts our previous expectation (16) that reannealing bias would lead amplicons originating from different templates to reach similar concentrations. A possible explanation for this discrepancy is the fact that each of the LH-PCR peaks represents SSU rDNAs originating from several different organisms. Reannealing between domains originating from different organisms could explain the observed shift in dominance between the two peaks. If the degree of similarity between the sequences with a domain A length of 317 bp were high enough for PCR amplicon cross-hybridization and kinetic inhibition, while the degree of similarity between sequences with a domain A length of 319 bp were low enough to not lead to cross-hybridization, one could envision that each of the 319-bp amplicons would experience lower levels of kinetic inhibition than the 317-bp amplicons. The average similarity among four SSU rDNA clones with a 317-bp domain A was 0.93 (0.89 to 1.00), while the degree of similarity between two SSU rDNA clones with a 319-bp domain A was 0.85, supporting this hypothesis. Another hypothesis which might explain the observed peak shift would be a large difference in the degree of diversity among the organisms corresponding to each of the peaks. Template reannealing inhibition should theoretically be lower for peaks with more gene types or peaks lacking a dominant gene type. This hypothesis cannot be tested with the data included in the current analysis.
Uncertainties about the numbers of ribosomal operons in different bacterioplankton species and differences in the relative sizes of genomes preclude the extrapolation from gene frequencies to cell abundance. Nevertheless, relative gene frequencies offer some advantages as a measurement for assessing the composition of natural microbial communities. In particular, rDNA frequency histograms (electropherograms) should be relatively insensitive to short-term variation in growth rates, which may affect rRNA abundance significantly under some circumstances (7).
LH-PCR is a promising method for the analysis of natural microbial populations. The main advantages of LH-PCR are that it surveys relative gene frequencies within complex mixtures of DNA, is reproducible, requires small sample sizes, and can be performed with many samples simultaneously. Furthermore, some of the size classes emerging from LH-PCR analyses can be related at the group level to environmental rDNA sequences. However, overlapping size classes leave ambiguities that require further analyses to resolve. The relative proportions of the electropherogram peaks from seawater are in agreement with previous findings that alpha-Proteobacteria members are dominant components of clone libraries constructed from DNA extracted from surface seawater. The observation that most SSU rDNAs of organisms cloned or cultivated from the same water sample have sizes that correspond to the peaks in the LH-PCR electropherograms also supports the validity of the method.
The main technical problem associated with LH-PCR is the accuracy of peak detection, especially when longer domains are used. Improvements in automated DNA sequencers and in Genescan software may increase the accuracy of the method for longer domains. This problem notwithstanding, domain B was useful to confirm the results obtained with domain A and, in some cases, to differentiate between phylogenetic groups with identical sizes in domain A, like the alpha-Proteobacteria and prymnesiophyte plastids.
The attributes of LH-PCR make it useful for quick assessments of the diversity of natural microbial communities for comparative purposes, for experimental designs that involve the manipulation of natural microbial communities (15), and for experiments, such as those we describe here, aimed at investigating the properties of PCR in applications employing complex mixtures of gene homologs.
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ACKNOWLEDGMENTS |
|---|
We are grateful to the staff of the Oregon State University Center for Gene Research Central Services Laboratory, and particularly to Anne-Marie Girard, for technical assistance.
This research was supported by grant OCE-9618530 from the National Science Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Oregon State University, Corvallis, OR 97331. Phone: (541) 737-1835. Fax: (541) 737-0496. E-mail: giovanns{at}bcc.orst.edu.
Present address: Monterey Bay Aquarium Research
Institute, Moss Landing, CA 95010.
Present address: Station Biologique, CNRS, INSU et
Université Pierre et Marie Curie, Roscoff Cedex, France.
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Top
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Discussion
References
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Applied and Environmental Microbiology, November 1998, p. 4522-4529, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Acinas, S. G., Sarma-Rupavtarm, R., Klepac-Ceraj, V., Polz, M. F.
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71: 8966-8969
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Paez-Rubio, T., Viau, E., Romero-Hernandez, S., Peccia, J.
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71: 804-810
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Drgon, T., Saito, K., Gillevet, P. M., Sikaroodi, M., Whitaker, B., Krupatkina, D. N., Argemi, F., Vasta, G. R.
(2005). Characterization of Ichthyocidal Activity of Pfiesteria piscicida: Dependence on the Dinospore Cell Density. Appl. Environ. Microbiol.
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Cardinale, M., Brusetti, L., Quatrini, P., Borin, S., Puglia, A. M., Rizzi, A., Zanardini, E., Sorlini, C., Corselli, C., Daffonchio, D.
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Coyne, K. J., Burkholder, J. M., Feldman, R. A., Hutchins, D. A., Cary, S. C.
(2004). Modified Serial Analysis of Gene Expression Method for Construction of Gene Expression Profiles of Microbial Eukaryotic Species. Appl. Environ. Microbiol.
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Yu, Z., Morrison, M.
(2004). Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol.
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McBain, A. J., Ledder, R. G., Moore, L. E., Catrenich, C. E., Gilbert, P.
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70: 3449-3456
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Jenkins, B. D., Steward, G. F., Short, S. M., Ward, B. B., Zehr, J. P.
(2004). Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray. Appl. Environ. Microbiol.
70: 1767-1776
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Sliwinski, M. K., Goodman, R. M.
(2004). Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling. Appl. Environ. Microbiol.
70: 1811-1820
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70: 214-223
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Rogers, G. B., Hart, C. A., Mason, J. R., Hughes, M., Walshaw, M. J., Bruce, K. D.
(2003). Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling. J. Clin. Microbiol.
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Egert, M., Friedrich, M. W.
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69: 2555-2562
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McBain, A. J., Bartolo, R. G., Catrenich, C. E., Charbonneau, D., Ledder, R. G., Rickard, A. H., Symmons, S. A., Gilbert, P.
(2003). Microbial Characterization of Biofilms in Domestic Drains and the Establishment of Stable Biofilm Microcosms. Appl. Environ. Microbiol.
69: 177-185
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Lueders, T., Friedrich, M. W.
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69: 320-326
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Curtis, T. P., Sloan, W. T., Scannell, J. W.
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Dunbar, J., Barns, S. M., Ticknor, L. O., Kuske, C. R.
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68: 3035-3045
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Frias-Lopez, J., Zerkle, A. L., Bonheyo, G. T., Fouke, B. W.
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Tiirola, M. A., Mannisto, M. K., Puhakka, J. A., Kulomaa, M. S.
(2002). Isolation and Characterization of Novosphingobium sp. Strain MT1, a Dominant Polychlorophenol-Degrading Strain in a Groundwater Bioremediation System. Appl. Environ. Microbiol.
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Zhou, J., Xia, B., Treves, D. S., Wu, L.-Y., Marsh, T. L., O'Neill, R. V., Palumbo, A. V., Tiedje, J. M.
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Small, J., Call, D. R., Brockman, F. J., Straub, T. M., Chandler, D. P.
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67: 4708-4716
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Friedrich, M. W., Schmitt-Wagner, D., Lueders, T., Brune, A.
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67: 4880-4890
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Diez, B., Pedros-Alio, C., Marsh, T. L., Massana, R.
(2001). Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques. Appl. Environ. Microbiol.
67: 2942-2951
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Madrid, V. M., Taylor, G. T., Scranton, M. I., Chistoserdov, A. Y.
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67: 1663-1674
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Qiu, X., Wu, L., Huang, H., McDonel, P. E., Palumbo, A. V., Tiedje, J. M., Zhou, J.
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Cottrell, M. T., Kirchman, D. L.
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Purkhold, U., Pommerening-Röser, A., Juretschko, S., Schmid, M. C., Koops, H.-P., Wagner, M.
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Suzuki, M. T., Taylor, L. T., DeLong, E. F.
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66: 4571-4574
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Lueders, T., Friedrich, M.
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Ritchie, N. J., Schutter, M. E., Dick, R. P., Myrold, D. D.
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