Comparisons of direct extraction methods of microbial DNA from different paddy soils

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.     

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.     

Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods

Simple and cost-effective methods are needed to extract DNA in order to use it in large-scale studies. Blood is an excellent DNA source; however, it is costly and invasive thus an alternative is needed. Several kits and chemical protocols using buccal cells have been proposed for DNA extraction. The objective of the study is to evaluate buccal NaOH chemical protocol and Nucleospin Tissue Kit (BD Biosciences, Macery-Nagel, Germany) for DNA extraction. DNA swab samples were collected from 300 voluntary participants. DNA yields and purity were measured by NaOH and Nucleospin Tissue Kit techniques; the cost and time consumption for DNA extraction per sample were assessed as well. Results have shown that DNA amount and purity extracted by NaOH procedure was compared to that of the kit (p = 0.164; p = 0.249, respectively). NaOH method was considered cheaper and less time consuming (0.06 versus 3.80 USD, and 1.33 versus 3.59 minutes per sample, p < 0.001). Buccal cell derived DNA extracted by NaOH protocol can be considered a feasible substitute for more expensive and time-consuming kits.     

Microwave-assisted extraction of glycyrrhizic acid from licorice root

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Optimisation of DNA extraction for AFLP analysis of mycorrhizal fungi of terrestrial orchids caladeniinae and drakaeinae

Published DNA extraction methods present a number of problems when applied to mycorrhizal fungi of native Australian terrestrial orchids. Grinding with liquid nitrogen shears the DNA, and other pulverisation methods yield too little DNA. We found that freezing the fungal sample with liquid nitrogen, with no grinding, followed by the Qiagen DNeasy extraction procedure produced good yields of high-molecular-weight DNA. The DNA was then used for amplified fragment length polymorphism (AFLP) fingerprinting. Good fingerprints were produced by restriction withEcoRI/MseI enzymes, the use of preamplification primer mix II (for small genomes), and a 2-base extensionMseI primer (m-cc) with 3-base extensionEcoRI primers in the selective amplification. This protocol may be of general utility for other fungi with similarly fragile DNA.     

Optimizing a continuous flow lipid extraction system (CFLES) used for extracting microalgal lipids

A laboratory‐made continuous flow lipid extraction system (CFLES) was devised to extract lipids from microalgae Nannochloropsis sp., a potential feedstock for biodiesel fuel, with a focus to assess the workable temperatures and pressures for future industrial applications. Using conventional solvents, the CFLES recovered 100% of the lipids extracted with conventional Soxhlet extraction. The optimum temperature and pressure were found to be 100 °C and 50 psi, respectively; conditions significantly lower than those normally used in pressurized liquid extractions requiring specialized equipment. Approximately 87% of the extracted oil was successfully transesterified into biodiesel fuel (fatty acid methyl esters). Preliminary calculations based on the tested lab‐scale system indicated savings in energy, solvent consumption, and extraction time as 96%, 80%, and more than 90%, respectively, as compared to Soxhlet extraction. However, the true cost savings can only be assessed at scaled up level. Energy efficiency of CFLES was calculated as 48.9%. Residual water (~70%) in the biomass had no effect on the extraction performance of CFLES, which is expected to help the process economics at scaled up application. The effect of temperature and pressure on the fatty acids profile of Nannochloropsis sp. is also discussed. Based on the existing literature, the authors believe that a pressurized liquid extraction system with continuous solvent flow has not been reported for lipid extraction from Nannochloropsis sp.     

Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils

DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

An effective DNA extraction protocol for brown algae

Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular‐based evolutionary studies. Our broad and long‐term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total‐DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total‐ DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques.     

Dehydration of a polyether type extraction agent and of the corresponding K+ complex: insights into liquid-liquid extraction mechanisms by quantum chemical methods

In this paper we report a quantum chemical study performed at the B3LYP/6-311G++(d,p) level of theory on structural and energetic aspects of the sequential dehydration of a tetra-hydrated polyethylene-glycol type podand (1,2-bis-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-benzene, hereafter b33) and its complex with the K(+) cation. Thermodynamical parameters were determined by hessian quantum calculations performed using a self-consistent reaction field (SCRF) method, taking into account solvent (dichloromethane) effects. The results allowed the estimation of dehydration enthalpies, entropies and free energies for the hydrated free b33 podand and its corresponding K(+) cation complex in dichloromethane. The low absolute values found for the dehydration free energies as well as the structural features found for the optimized structures and the corresponding basis superposition calculated interaction energies, support the hypothesis of an interfacial complexation type mechanism governing the assisted extraction of K(+) from an aqueous toward an organic phase, in liquid/liquid extraction.     

A high-throughput plant DNA extraction method for marker analysis

The use of molecular markers to improve crops depends on the availability of rapid and efficient DNA extraction methods. Here we describe a simple and inexpensive method to isolate plant DNA suitable for RFLP, AFLP, and simple sequence repeat (SSR) analysis. This procedure uses stainless steel ball bearings to grind 16 samples simultaneously using a high-speed flask shaker. The method used in routine laboratory exercises yields 120–144 DNA extractions in a day by a single person at a cost of $0.60 (AUD) per sample, doubling the throughput of conventional methods.     

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves

Journal of Plant Research - Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of...     

An optimized method for the extraction of ancient eukaryote DNA from marine sediments

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo‐communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead‐beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica‐solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size‐selection of low molecular‐weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead‐beating, DNA binding in silica‐solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post‐library LMW size‐selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data‐processing protocol should improve quantitative paleo‐monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Predicting efficiency of different chemical extraction methods in risk assessment of trace metals in sediment of the Persian Gulf

Different chemical methods have been developed to evaluate the bioavailable fraction of the trace metals. Due to the lack of a universal method for analysis of the bioavailable metal fractions, due to the differences in sediment characteristics, it is necessary to validate an appropriate chemical method for assessing the available fraction of trace metals. For this propose, in this study, different chemical extraction methods including extraction with HCl and desorption test as a single reagent leaching test as well as geochemical fractions method have been evaluated. Bushehr coastal sediments in the Persian Gulf coasts have been selected for this purpose. To validate the efficacy of these methods, a gastropod species (Trochus erithreus), as a bioindicator, has been selected and monitored for trace metals of Fe²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Mn²⁺, and Ni²⁺. According to the one-way ANOVA results, all partial extraction methods for all the trace metals (except for Mn²⁺) showed no significant variation, but linear correlation coefficients were between the results of the selective chemical extraction methods and concentrations of trace metals in organism tissues. It offered that selective extraction with HCl can be used as a simple method for measuring the bioavailable fraction of the metals in the sediments.