scholarly journals Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

2005 ◽  
Vol 71 (8) ◽  
pp. 4721-4727 ◽  
Author(s):  
Stefan J. Green ◽  
Dror Minz
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dna Templates ◽  
Target Dna ◽  
Gradient Gel Electrophoresis ◽  
Pcr Method

ABSTRACT PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.

Changes in the structure and diversity of bacterial communities during the process of adaptation to organic wastewater

2010 ◽  
Vol 56 (4) ◽  
pp. 352-355 ◽  
Author(s):  
Junmin Li ◽  
Zexin Jin ◽  
Binbin Yu
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Similarity Index ◽  
Pcr Amplification ◽  
Genotypic Diversity ◽  
Diversity Indices ◽  
Inhibitory Effect ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis

To explore changes in the structure and diversity of activated sludge-derived microbial communities during adaptation to gradual increases in the concentration of wastewater, RAPD–PCR and the combination of PCR amplification of 16S rRNA genes with denaturing gradient gel electrophoresis (DGGE) analysis were used. In bacterial communities exposed to 0%, 5%, 10%, 20%, or 40% wastewater, there were 27, 25, 18, 17 and 16 bands, respectively, based on DGGE data, while there were 69, 83, 97, 86, and 88 bands, respectively, based on RAPD data. The community similarity index among bacterial communities during the process of adaptation to different concentrations of wastewater was different based on DGGE and RAPD data. Based on DGGE and RAPD profiles, the Shannon–Weiner and Simpson’s diversity indices decreased sharply upon exposure to 10% wastewater, indicating that 10% wastewater might be a critical point at which the growth of bacteria could be significantly inhibited and the genotypic diversity could change. This indicated that changes in structure and diversity might have an inhibitory effect on the toxicity of organic matter and that selection and adaptation could play important roles in the changes.

Genetic Diversity of the Biofilm CoveringMontacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

1998 ◽  
Vol 64 (9) ◽  
pp. 3464-3472 ◽  
Author(s):  
David C. Gillan ◽  
Arjen G. C. L. Speksnijder ◽  
Gabriel Zwart ◽  
Chantal De Ridder
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Dna Extraction ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis ◽  
Biofilm Bacteria ◽  
Sequence Types

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas,Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of theCytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

scholarly journals Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

2000 ◽  
Vol 66 (10) ◽  
pp. 4372-4377 ◽  
Author(s):  
Bo Normander ◽  
Jim I. Prosser
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Populations ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

ABSTRACT An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

2002 ◽  
Vol 48 (4) ◽  
pp. 333-341 ◽  
Author(s):  
Christine A Morgan ◽  
Andre Hudson ◽  
Allan Konopka ◽  
Cindy H Nakatsu
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Community Response ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Specific Content ◽  
Gradient Gel Electrophoresis

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: α, β, and γ Proteobacteria and the high GC and the low GC Gram-positive divisions.Key words: 16S rRNA, DGGE, community analysis, biomass-recycle reactor.

scholarly journals Characterization of the Prokaryotic Diversity in Cold Saline Perennial Springs of the Canadian High Arctic

2007 ◽  
Vol 73 (5) ◽  
pp. 1532-1543 ◽  
Author(s):  
Nancy N. Perreault ◽  
Dale T. Andersen ◽  
Wayne H. Pollard ◽  
Charles W. Greer ◽  
Lyle G. Whyte
Keyword(s):  
16S Rrna ◽  
Clone Library ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Geographic Origin ◽  
High Arctic ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Cold Environments ◽  
Gradient Gel Electrophoresis ◽  
High Concentrations

ABSTRACT The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of −325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.

scholarly journals Development and Application of a Selective PCR-Denaturing Gradient Gel Electrophoresis Approach To Detect a Recently Cultivated Bacillus Group Predominant in Soil

2004 ◽  
Vol 70 (10) ◽  
pp. 5801-5809 ◽  
Author(s):  
Vesela A. Tzeneva ◽  
Youguo Li ◽  
Andreas D. M. Felske ◽  
Willem M. de Vos ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
16S Rrna ◽  
The Netherlands ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Physiological State ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

ABSTRACT The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world.

scholarly journals Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

2007 ◽  
Vol 74 (3) ◽  
pp. 889-893 ◽  
Author(s):  
Zhongtang Yu ◽  
Rubén García-González ◽  
Floyd L. Schanbacher ◽  
Mark Morrison
Keyword(s):  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
V Region

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

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