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A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes.

The article describes a method for extracting DNA from soil which involves minimal purification prior to polymerase chain reaction (PCR) amplification. A fundamental obstacle to understanding microbial ecology and diversity is the inability to culture microorganisms from environmental samples. Extraction methods investigated include sonication, enzymatic lysis and extraction from bacterial cells isolated from soil. A comparison of extraction methods using a single soil collected from Macquarie University is presented.

Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3-45 % and decreased the protein contamination by 22.5-34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA. [ABSTRACT FROM AUTHOR]

Talc (hydrated magnesium silicate) is a physical coadjuvant that can be employed in the production of extra virgin olive oil to increase yield. The adsorbent properties of talc could hamper DNA recovery, leading to false negatives in DNA analysis. The aim of this work was to verify the effect of talc addition on olive oil DNA by targeting four selected microsatellites. Olive processing trials were carried out at two different levels of talc (1 and 2%) and without talc (control). DNA extraction yield and purity level were ascertained, and High Resolution Melting (HRM) analysis of microsatellites was subsequently applied to the extracted DNA. Neither the DNA extraction yield nor the A260/230 and A260/280 ratios showed significant differences between control and talc-treated samples. Higher values of A260/230 and lower yields were observed in olive oils than in olive pastes. The DNA microsatellites analyzed showed identical HRM profiles in all the samples, excluding any effect of talc and confirming the genetic homogeneity of the olive lot processed (cv. Coratina). [ABSTRACT FROM AUTHOR]

Aims The development of high-throughput methods such as pyrosequencing and microarrays has greatly improved our understanding of the microbial diversity in complex environments such as soils. Nevertheless, albeit advancements in such techniques, the first major step is to obtain high quantity and good quality genomic DNA (g DNA). The work presented here aims to present an inherent problem with 260 : 230 nm ratio of extracted g DNA from calcareous soils of Tuber melanosporum orchards and a protocol to overcome this problem. Methods and Results Using two commercial g DNA extraction kits on spatially distant truffle orchards, we demonstrated that the 260 : 230 nm ratio was very low, consequentially yielding g DNA incompatible with microarray analyses. In order to solve this problem, optimization steps were tested including several wash steps performed before and/or after lysis. These washes significantly improved the g DNA quality (ratio 260 : 230 nm >1·7) without modification of the structure of the bacterial communities as stated by temporal temperature gradient gel electrophoresis analysis. A final re-extraction with phenol/chloroform was required for one of the soil samples. Conclusions A combination of wash steps included into the extraction protocol followed by phenol: chloroform re-extraction is recommended to obtain high-quality g DNA from calcareous soils of T. melanosporum orchards. Significance and Impact of the Study The method recommended here significantly improves g DNA quality obtained from T. melanosporum orchards to make it acceptable for highly sensitive methods such as microarray. [ABSTRACT FROM AUTHOR]

Background and Aims Bunch rot symptoms can appear weeks after the infection of grape flowers by Botrytis cinerea. Quantitative polymerase chain reaction ( qPCR) detects changes in the DNA mass of a target organism and is a potential tool for studying quiescent infections. The aim was to optimise a duplex qPCR to quantify B. cinerea DNA in the background of endogenous Vitis vinifera DNA. Methods and Results Three DNA extraction techniques and three probe sets were compared. The optimised qPCR using the Bc3 probe set was 1000-fold more sensitive than other probe sets, with a threshold cycle value of <33 for as little as 1 picogram of B. cinerea DNA. The duplex assay successfully detected an increasing amount of B. cinerea DNA when mixed with V. vinifera DNA or of B. cinerea conidia when added to grape receptacles. Conclusions Duplex assays quantifying B. cinerea DNA in the background of endogenous grape DNA were efficient and sensitive, with calculation of a pathogen coefficient allowing comparison of results among assays. Significance of the Study The results demonstrate the potential to monitor symptomless, quiescent infections and to investigate the consequence of an intervention (e.g. a fungicide treatment) before disease symptoms are visible. [ABSTRACT FROM AUTHOR]

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Objectives: To assess the genomic DNA from marine soil sediment for construction metagenomic library. Methods: The soil samples were collected from the study area at coastal regions of Samiyarpettai, Cuddalore District, TamilNadu,(south India),during the month of April 2010. Genomic DNA was extracted for marine soil sediment by the method of direct isolation (nature protocol), purification of DNA is followed by the chromous gel extraction kits. Results: Approximately 6-7 mg of genomic DNA extract was derived for construction of metagenomic library, and also high quality DNA was derived by the elctroelution purification process. Conclusion: From this study it is confirmed that the direct isolation of DNA method is an efficient protocol for isolation of high amount and good quality of genomic DNA for the construction of metagenomic library. The elctroelution (Chromous Gel Extraction kits) method is an effective for purifying the genomic DNA and also to get rid off humic substances and other contaminants when compare to various purification kits. [ABSTRACT FROM AUTHOR]

In many areas of the world, polybrominated diphenyl ethers are ubiquitous due to their use as fire retardants. It is known that the hydrophobic characteristics of PBDEs cause them to sink in soil and sediment, yet their effect on microbes within the soil is not well understood. In this study, soil was treated with 1, 10, and 100 mg kg BDE 209 for up to 45 days. Treatment effects on soil enzymatic activities for urease and catalase were evaluated. The impact on the microbe community structure was estimated using denaturing gradient gel electrophoresis after polymerase chain reaction amplification of total genomic DNA, using bacterial variable V3 region targeted primers. The effects on the soil microbial community size and major bacterial groups were evaluated using fluorescence in situ hybridization analysis. Forty-five days after the addition of BDE 209, urease activity was suppressed by BDE 209, even at a concentration of 1 mg kg. Catalase activity increased in the samples containing lower concentrations of BDE 209, but was suppressed in samples containing higher concentrations. The bacterial community also varied in response to the addition of BDE 209, and the variation of community composition differed among treatments. In addition, α, β and γ subclass proteobacteria decreased in the group of 100 mg kg BDE 209 spiked soil after 45 days of treatment. Throughout the experiment, no BDE 209 degradation was detected under darkness. These observations demonstrated that BDE 209 in soil, although of low bioavailability, had an adverse impact on the structure and function of the soil microbial community and microbial processes. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluates the DNA extraction methods for various soil samples including indirect method and alkaline lysis method. The study compared the methods' spectrophotometric quality, DNA yield, polymerase chain reaction (PCR) suitability, and genomic integrity. It reveals that the DNA obtained through alkaline lysis method was the best. It notes that alkaline lysis method is a versatile, reliable, and novel technique for large-scale extraction of DNA.

Background: With the rapid development of genome sequencing techniques, traditional research methods based on the isolation and cultivation of microorganisms are being gradually replaced by metagenomics, which is also known as environmental genomics. The first step, which is still a major bottleneck, of metagenomics is the taxonomic characterization of DNA fragments (reads) resulting from sequencing a sample of mixed species. This step is usually referred as "binning". Existing binning methods are based on supervised or semi-supervised approaches which rely heavily on reference genomes of known microorganisms and phylogenetic marker genes. Due to the limited availability of reference genomes and the bias and instability of marker genes, existing binning methods may not be applicable in many cases. Results: In this paper, we present an unsupervised binning method based on the distribution of a carefully selected set of l-mers (substrings of length l in DNA fragments). From our experiments, we show that our method can accurately bin DNA fragments with various lengths and relative species abundance ratios without using any reference and training datasets. Another feature of our method is its error robustness. The binning accuracy decreases by less than 1% when the sequencing error rate increases from 0% to 5%. Note that the typical sequencing error rate of existing commercial sequencing platforms is less than 2%. Conclusions: We provide a new and effective tool to solve the metagenome binning problem without using any reference datasets or markers information of any known reference genomes (species). The source code of our software tool, the reference genomes of the species for generating the test datasets and the corresponding test datasets are available at http://i.cs.hku.hk/~alse/MetaCluster/. [ABSTRACT FROM AUTHOR]

Background: It has been shown that integron-associated gene cassettes exist largely in tandem arrays of variable size, ranging from antibiotic resistance arrays of three to five cassettes up to arrays of more than 100 cassettes associated with the vibrios. Further, the ecology of the integron/gene cassette system has been investigated by showing that very many different cassettes are present in even small environmental samples. In this study, we seek to extend the ecological perspective on the integron/gene cassette system by investigating the way in which this diverse cassette metagenome is apportioned amongst prokaryote lineages in a natural environment. Results: We used a combination of PCR-based techniques applied to environmental DNA samples and ecological analytical techniques to establish co-assortment within cassette populations, then establishing the relationship between this co-assortment and genomic structures. We then assessed the distribution of gene cassettes within the environment and found that the majority of gene cassettes existed in large co-assorting groups. Conclusions: Our results suggested that the gene cassette diversity of a relatively pristine sampling environment was structured into co-assorting groups, predominantly containing large numbers of cassettes per group. These co-assorting groups consisted of different gene cassettes in stoichiometric relationship. Conservatively, we then attributed co-assorting cassettes to the gene cassette complements of single prokaryote lineages and by implication, to large integron-associated arrays. The prevalence of large arrays in the environment raises new questions about the assembly, maintenance and utility of large cassette arrays in prokaryote populations. [ABSTRACT FROM AUTHOR]

Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene ( benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene ( benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches. [ABSTRACT FROM AUTHOR]

A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar > geographical site > harvest period > growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves. [ABSTRACT FROM AUTHOR]

Class 1 integrons are an important vector for the spread of antibiotic resistance. The core of this genetic element is highly conserved in all class 1 integrons recovered from clinical contexts. Recently, bacteria containing more divergent class 1 integrons have been isolated from environmental samples, suggesting undiscovered diversity in these elements. We performed a culture-independent survey of the class 1 integron-integrase gene ( intI1) from environmental DNA, assessing sequence variation using capillary electrophoresis single-strand conformation polymorphism. This analysis allowed informed selection of environments for further investigation based on the diversity of intI1 targets that were present. IntI1 was common in environmental samples and exhibited previously unsuspected sequence diversity. The method allowed discrimination between clinical and environmental variants of intI1. [ABSTRACT FROM AUTHOR]

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica-magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR-ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation. [ABSTRACT FROM AUTHOR]

A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. For Myrothecium gramineum, the highest DNA concentration was obtained with the procedure described by N. Vanittanakom et al. (J Clin Microbiol 2002, 40: 1739–1742). For A. nidulans, concentrations higher than 100 ng/μl were reached with the glass bead, the LiCl, the boiling, the liquid N2 and the protoplast-based method. Samples of M. gramineum resulting from the boiling and the liquid N2 procedure were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb. Amplification of fragments up to 4.3 kb requires the use of the Elongase Mix on samples extracted with the liquid N2 procedure. [ABSTRACT FROM AUTHOR]

The effectiveness of four soil DNA extraction methods for total microbial DNA isolation from five types of soils was compared. These methods were evaluated comprehensively by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) analysis of the bacterial community. The results showed that the sodium dodecyl sulphate (SDS)–high strength salt method gave the highest soil DNA yield and the DNA Extraction Kit method was better for DNA purification. Following PCR-DGGE analysis, the DNA extracted by the DNA Extraction Kit method presented a more diverse bacterial community than others, whereas the SDS-phenol–chloroform method provided the poorest bacterial community diversity. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5 × 10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability. [ABSTRACT FROM AUTHOR]

Phylogeographic analyses of the fauna of the Australian wet tropics rainforest have provided strong evidence for long-term isolation of populations among allopatric refugia, yet typically there is no corresponding divergence in morphology. This system provides an opportunity to examine the consequences of geographic isolation, independent of morphological divergence, and thus to assess the broader significance of historical subdivisions revealed through mitochondrial DNA phylogeography. We have located and characterized a zone of secondary contact between two long isolated (mtDNA divergence > 15%) lineages of the skink Carlia rubrigularis using one mitochondrial and eight nuclear (two intron, six microsatellite) markers. This revealed a remarkably narrow (width < 3 km) hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes at two of three nuclear loci with diagnostic alleles. Cline centers were coincident across loci. Using a novel form of likelihood analysis, we were unable to distinguish between sigmoidal and stepped cline shapes except at one nuclear locus for which the latter was inferred. Given estimated dispersal rates of 90-133 m x gen-1/2 and assuming equilibrium, the observed cline widths suggest effective selection against heterozygotes of at least 22-49% and possibly as high as 70%. These observations reveal substantial postmating isolation, although the absence of consistent deviations from Hardy-Weinberg equilibrium at diagnostic loci suggests that there is little accompanying premating isolation. The tight geographic correspondence between transitions in mtDNA and those for nuclear genes and corresponding evidence for selection against hybrids indicates that these morphologically cryptic phylogroups could be considered as incipient species. Nonetheless, we caution against the use of mtDNA phylogeography as a sole criterion for defining species boundaries. [ABSTRACT FROM AUTHOR]

PCR methods have been shown to be biased by several factors. In the present study, we have developed a theoretic and practical approximation to elucidate how the presence of mismatches at the primers annealing regions and the different number of rDNA operons per cell can influence PCR and subsequent restriction fragment length polymorphism (RFLP) analyses from bacterial populations. We have performed RFLP analyses of 16S rRNA genes amplified by PCR from mixed bacterial cultures showing different primer identities and number of rDNA operons. Our results clearly corroborate that both factors, number of rDNA operons and primers identity, clearly influence the 16S rDNA-RFLP estimations. It has been demonstrated that a higher number of operons leads to a higher efficiency of detection, but a lower degree of primer complementarity implies a decrease in such efficiency. [ABSTRACT FROM AUTHOR]

There are an increasing number of assays available for fungal plant pathogens based on DNA technology. We have developed such an assay for Gaeumannomyces graminis var. tritici (Ggt) in soil, using slot-blot hybridisation. To ensure the validity of DNA-based soil assays for the fungus, it is important to determine the stability of Ggt DNA in soil. This study was undertaken to quantify the DNA degradation of dead Ggt in soil using a DNA-based assay. Mycelia were killed using various treatments, then DNA was extracted and estimated by a slot-blot hybridisation technique using the specific Ggt DNA probe, pG158. Mycelia were also killed using a fungicide (triadimefon) at a concentration of 150–250 μg ml−1. The amount of detectable DNA of Ggt, killed using triadimefon, declined by 82–93%. Inoculum in the form of diseased wheat roots, artificially inoculated ryegrass seed, particulate soil organic matter and whole soil was killed using heat-treatment. The amount of detectable DNA of Ggt declined markedly (90%) in both heat-treated roots and inoculated ryegrass seeds, and declined by 50% in both treated soil and soil organic matter. The rate of DNA degradation of Ggt in soil varied with the type of inoculum. The amount of detectable DNA of Ggt in dead mycelia declined by 99.8% after 4 days of incubation in soil. No DNA was detected after 8 days of incubation. In contrast, Ggt DNA in live mycelia declined by 70% after 8 days of incubation and declined to 10% of original DNA level after 32 days. In ground ryegrass seed inoculum, DNA in both killed and live Ggt declined by 50% after 8 days. In diseased roots, DNA from both live and killed Ggt did not appear to decline over 16 days. Estimates of the amount of Ggt in the soil using a DNA-based assay reflect both live and dead populations of the fungus. The rate of breakdown of DNA of the dead fungus is very high and the presence of dead fungi in roots probably a rare event so the DNA from dead fungus probably contributes little to the total DNA level. [Copyright &y& Elsevier]

Presents a study which examined the isolation of DNA from forest soil suitable for polymerase chain reaction assays of fungal and plant rRNA genes. Uses of the obtained DNA; Review of related literature; Analysis of DNA and amplification products; Steps required to obtain good genomic material.

As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology. [ABSTRACT FROM AUTHOR]