scholarly journals Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil

2010 ◽  
Vol 76 (10) ◽  
pp. 3378-3382 ◽  
Author(s):  
Özgül İnceoǧlu ◽  
Eelco F. Hoogwout ◽  
Patrick Hill ◽  
Jan Dirk van Elsas
Keyword(s):  
Extraction Method ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Agricultural Soils ◽  
Extraction Methods ◽  
The Novel ◽  
Soil Dna ◽  
Dna Extraction Method ◽  
Gradient Gel Electrophoresis ◽  
Novel Method ◽  
Microbial Groups

ABSTRACT Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods. Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.

Archaeal ammonia oxidisers are abundant in acidic, coarse-textured Australian soils

Soil Research ◽  
2011 ◽  
Vol 49 (8) ◽  
pp. 715 ◽  
Author(s):  
Cathryn A. O'Sullivan ◽  
Steven A. Wakelin ◽  
Ian R. P. Fillery ◽  
Adrienne L. Gregg ◽  
Margaret M. Roper
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Agricultural Soils ◽  
Amoa Gene ◽  
Banding Patterns ◽  
Soil Dna ◽  
Potential Nitrification ◽  
Gene Copies ◽  
Gradient Gel Electrophoresis ◽  
Key Driver ◽  
Ammonia Oxidising Archaea

The abundances of ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soils underlying pastures in the south-west of Western Australia (WA) were investigated. Samples were collected from irrigated pastures and one unmanaged (driveway) area during December 2009. Archaeal and bacterial ammonia monooxygenase (amoA) genes were quantified using real-time PCR, and the diversity of the archaeal amoA genes was investigated using denaturing gradient gel electrophoresis (PCR-DGGE). AOA amoA gene copies outnumbered AOB in all samples. Numbers of archaeal amoA genes ranged from 4.1E+01 to 1.34E+05 gene copies/ng soil DNA. Bacterial amoA genes were below detection limits at three of the four sample sites and ranged from 8.9E+01 to 6.7E+02 gene copies/ng soil DNA at the remaining site. Potential nitrification rates (PNR) were not correlated with AOA or AOB gene abundance, but high PNR only occurred at the site with measureable numbers of AOB. The DGGE analysis revealed that the AOA community was diverse and variability in banding patterns was significantly affected by both site and depth (P < 0.05). Statistical analysis matching biological variation (AOA amoA genotypes) to environmental variables (BEST analysis) revealed that pH was the key driver of AOA community structure (ρ = 0.72; P = 0.005). Soil pH was also inversely correlated to abundance of AOA amoA genes in soil (ρ = 0.8; P = 0.003). This study has shown that AOA are important members of the nitrogen-cycling community in acidic WA pasture soils, and likely in the wider agricultural soils of WA.

scholarly journals An Improved Procedure of the Metagenomic DNA Extraction from Saline Soil, Sediment and Salt

2016 ◽  
Vol 60 ◽  
pp. 38-45
Author(s):  
Fereshteh Jookar Kashi
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Large Scale ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Skim Milk ◽  
Community Analysis ◽  
Functional Genes ◽  
Dna Extraction Method ◽  
Gradient Gel Electrophoresis ◽  
Direct Dna Extraction

A new modified protocol has been developed for extracting pure community inhibitors-free DNA from saline soils, sediments and salts. Amplification of DNA from soil and sediment is often inhibited by copurified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to saline samples. Using a widely used a newly modified direct DNA extraction method proposed in this report, DNA was extracted from samples of Urmia Lake in diverse geological location in Iran and quantity of the DNA were examined. We developed an improved method to extract DNA include the combination of physical, chemical and mechanical lysis methods from saline samples. In the earlier reports, skim milk as an adsorption competitor was added to buffer DNA extract. In current study, we added skim milk to buffer DNA extraction. The results showed that skim milk was useful as an additive for extract DNA from saline samples. This method is applicable to molecular community analysis of saline samples which strongly adsorb DNA. The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA. The extracted DNA was used to successfully amplify 16SrRNA region and functional genes. The amplicons were suitable for further applications such as diversity based analysis by denaturing gradient gel electrophoresis (DGGE) and cloning library.

scholarly journals Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

2006 ◽  
Vol 72 (10) ◽  
pp. 6452-6460 ◽  
Author(s):  
Paul J. Hunter ◽  
Geoff M. Petch ◽  
Leo A. Calvo-Bado ◽  
Tim R. Pettitt ◽  
Nick R. Parsons ◽  
...  
Keyword(s):  
Microbial Activity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Small Subunit ◽  
Disease Suppression ◽  
Rrna Gene ◽  
Damping Off ◽  
Small Subunit Rrna ◽  
Pythium Sylvaticum ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

scholarly journals Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

2020 ◽  
Vol 5 (2) ◽  
pp. 95 ◽  
Author(s):  
Rajashree Chowdhury ◽  
Prakash Ghosh ◽  
Md. Anik Ashfaq Khan ◽  
Faria Hossain ◽  
Khaledul Faisal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Recombinase Polymerase Amplification ◽  
National Guidelines ◽  
Diagnostic Efficacy ◽  
Rapid Extraction ◽  
Kala Azar ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

scholarly journals Evaluation of Biochar Nitrate Extraction Methods

2019 ◽  
Vol 9 (17) ◽  
pp. 3514 ◽  
Author(s):  
Jenna Walsh ◽  
Joseph Sanford ◽  
Rebecca Larson
Keyword(s):  
Extraction Method ◽  
Extraction Efficiency ◽  
Agricultural Soils ◽  
Extraction Methods ◽  
Multiple Methods ◽  
Soil Extraction ◽  
Future Studies ◽  
Different Types ◽  
Increasing Temperature ◽  
Biochar Amendment

Biochar amendment to soil is a method used to mitigate losses of nitrogen leaching through agricultural soils. Multiple methods for extraction of nitrogen have been used, and recent studies have indicated that traditional soil extraction methods underestimate biochar nitrate. This study evaluated the nitrate extraction efficiency of a KCl extraction method under different temperature (20 and 50 °C) and duration (24 and 96 h) conditions. Increasing the duration of extraction from 24 to 96 h did not have a significant impact on extraction efficiency. However, increasing temperature resulted in nitrate extraction efficiencies above 90%. Rinsing the biochar once with deionized (DI) water following filtration after extraction increased the extraction efficiency significantly, but any subsequent rinses were not significant. This study recommends extracting nitrate from biochar using 2 M KCl at 50 °C for a period of 24 h with one additional rinse to increase nitrate recovery above 90%. However, future studies should evaluate this procedure for different types of biochar produced from alternative biomasses and at varying temperatures.

Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway) - denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures

2003 ◽  
Vol 49 (10) ◽  
pp. 602-612 ◽  
Author(s):  
Ingvild Wartiainen ◽  
Anne Grethe Hestnes ◽  
Mette M Svenning
Keyword(s):  
Methane Emission ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
The Arctic ◽  
Rrna Gene ◽  
Climatic Warming ◽  
Soil Dna ◽  
Enrichment Cultures ◽  
Arctic Soil ◽  
Gradient Gel Electrophoresis

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78°N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 °C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.Key words: methanotrophic diversity, Svalbard, arctic wetland, denaturing gradient gel electrophoresis.

Isolation and purification of microbial community DNA from soil naturally enriched for chitin

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Pullabhotla Sarma ◽  
Vadlamudi Srinivas ◽  
Kondreddy Anil ◽  
Appa Podile
Keyword(s):  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sodium Borate ◽  
Metagenomic Dna ◽  
Soil Dna ◽  
Isolation And Purification ◽  
Gradient Gel Electrophoresis ◽  
Chitinase Genes ◽  
Sodium Borate Buffer

AbstractWe made an attempt to isolate and purify metagenomic DNA from chitin enriched soil. In this communication we report a modified direct lysis method for soil DNA extraction including initial pre-lysis washing of sample, followed by a rapid polyvinylpyrrolidone-agarose-based purification and electroelution of DNA using Gene-capsule™ assembly. Rapidity was achieved using low molarity conducting media (sodium-borate buffer) for electrophoresis by reducing run time for both the gel electrophoresis and electroelution. Extracted DNA was sufficiently pure and of high quality, evidenced by amplification of 16S rDNA and chitinase genes by PCR. Metagenomic nature of the DNA was confirmed by running V3 (16S rDNA) region amplicons using denaturing gradient gel electrophoresis. This method requires 30 min for purification, and less than 2 h for complete execution of protocol and becomes the first report on the isolation of metagenomic DNA from soil naturally enriched for chitin.

Characterization of bacterial and fungal communities in composted biosolids over a 2 year period using denaturing gradient gel electrophoresis

2009 ◽  
Vol 55 (4) ◽  
pp. 375-387 ◽  
Author(s):  
Amy Novinscak ◽  
Nadine J. DeCoste ◽  
Céline Surette ◽  
Martin Filion
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Fungal Species ◽  
Fungal Communities ◽  
Whole Process ◽  
Agricultural Applications ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups ◽  
Key Microorganisms

Composting is a microbial process that converts organic waste into a nutrient-rich end product used in horticultural and agricultural applications. The diversity and long-term succession of microorganisms found in composted biosolids has been less characterized than other composts. In this study, bacterial and fungal communities found in composted biosolids aging from 1 to 24 months were studied using denaturing gradient gel electrophoresis (DGGE) and sequencing. The results revealed high levels of diversity, where 53 bacterial species belonging to 10 phyla and 21 fungal species belonging to 4 phyla were identified. Significant differences were observed when comparing the bacterial DGGE patterns of young compost samples, whereas no differences were observed in samples over 8 months. For fungal patterns, no significant differences were observed during the first 4 months of composting, but the diversity then significantly shifted until 24 months. The results indicate that patterns of bacterial species vary during the first few months of composting, whereas fungal patterns generally vary throughout the whole process, except during early stages. The description of the main microbial groups found in composted biosolids could find various applications, including the discovery of biotechnologically relevant microorganisms and the development of novel markers allowing quantitative monitoring of key microorganisms.

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