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.     

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.     

Genomic DNA extraction from<Emphasis Type="Italic">Victoria amazonica</Emphasis>

DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP) (Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A_260/230 ratio of 1.836, A_260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion.     

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.     

东方白鹳粪便总DNA五种提取方法的比较

本研究比较东方白鹳(Ciconia boyciana)粪便总DNA的5种提取方法,旨在为后续物种性别鉴定和DNA条形码鉴定提供合适的方法.采用十六烷基三甲基溴化铵法(CTAB法)、十二烷基磺酸钠裂解法(SDS法)、Tiangen试剂盒法、Qiagen试剂盒法和异硫氰酸胍法(GuSCN法)对取自天津动物园的东方白鹳新鲜粪...     

Optimization of the extraction and purification of the compatible solute ectoine from <Emphasis Type="Italic">Halomonas elongate</Emphasis> in the laboratory experiment of a commercial production project

Optimization of compatible solutes (ectoine) extraction and purification from Halomonas elongata cell fermentation had been investigated in the laboratory tests of a large scale commercial production project. After culturing H. elongata cells in developed medium at 28?°C for 23–30 h, we obtained an average yield and biomass of ectoine for 15.9 g/L and 92.9 (OD₆₀₀), respectively. Cell lysis was performed with acid treatment at moderate high temperature (60–70?°C). The downstream processing operations were designed to be as follows: filtration, desalination, cation exchange, extraction of crude product and three times of refining. Among which the cation exchange and extraction of crude product acquired a high average recovery rate of 95 and 96%; whereas a great loss rate of 19 and 15% was observed during the filtration and desalination, respectively. Combined with the recovering of ectoine from the mother liquor of the three times refining, the average of overall yield (referring to the amount of ectoine synthesized in cells) and purity of final product obtained were 43% and over 98%, respectively. However, key factors that affected the production efficiency were not yields but the time used in the extraction of crude product, involving the crystallization step from water, which spended 24–72 h according to the production scale. Although regarding to the productivity and simplicity on laboratory scale, the method described here can not compete with other investigations, in this study we acquired higher purity of ectoine and provided downstream processes that are capable of operating on industrial scale.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

T-lymphocyte mediated cytolysis: Temperature dependence of killer cell dependent and independent phases and lack of recovery from the lethal hit at low temperatures

Using alloantiserum and complement to inactivate cytolytic T-lymphocytes after they had administered the “lethal hit” to target cells, the rate of killer-cell independent lysis (KCIL) as measured by radiochromium release was followed at various temperatures. Under usual conditions, KCIL was half-completed on the average after 1.7 hr at 37 °C. The average Q₁₀ of KCIL is about 1.6 during the first few hours after cooling, but near 0 °, lysis slows down at later times. Thus, the extent of KCIL after 6–8 hr at 0 ° is frequently less than one-tenth of that at 37 °C. The Q₁₀ of the whole killing process is 2.5 near 37 °C but exceeds 6 near 22 °C.Evidence has been presented elsewhere suggesting that recovery from complement mediated damage may occur under appropriate conditions. Since KCIL can largely be arrested at low temperatures, we tested for possible recovery from or repair of the T-cell administered “lethal hit” during incubations at low temperature following (i) inactivation of killer cells by antiserum and complement or (ii) detachment of killer cells with EDTA and prevention of subsequent killer-target cell contact with dextran. No evidence for recovery from the “lethal hit” was found during incubations from 0.3 to 5 hr at 20 °, 15 °, or 0 °C. The temperature dependence of KCIL raises the possibility that metabolic events are of importance during KCIL. However, the previous finding that lysis following damage mediated by antiserum and complement is equally temperature sensitive leaves no basis for postulating such metabolic events. Hence, although unequivocal direct evidence has been difficult to obtain, colloid osmotic lysis is at present the simplest and most plausible explanation of killer-cell independent lysis.     

DNA recovery from soils of diverse composition.

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.     

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze–thaw–SDS–Protein K) and FTSPP (Freeze–thaw–SDS–Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL^?1 BSA. However, the FTSPP extraction method with DNA purification by a Wizard^® kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.     

Reproducibility and correlation study of three deoxyribonucleic acid hybridization procedures

Reproducibility of relative binding ratios (RBR) was studied with 26 labeled DNA-unlabeled DNA hybridization reactions carried out at two temperatures (60 and 75°C) and following three procedures: the S1-nuclease method with precipitation of S1-resistant DNA by trichloroacetic acid (S1-TCA), the S1-nuclease method with adsorption of S1-resistant DNA onto DE81 filters (S1-DE81), and the hydroxyapatite (HA) method. Analyses of variance indicated that different hybridization methods give different RBR, but similar percent divirgence (ΔT _m) results. A curvilinear relationship was found between RBR data obtained with S1-TCA and HA procedures at 60 or 75°C, and between 60°C RBR data obtained with S1-DE81 and HA procedures. At 75°C, RBR obtained with S1-DE81 and HA methods are comparable without any transformation of the data.     

Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Chemical Extraction of Arsenic from Contaminated Soils in the Vicinity of Abandoned Mines and a Smelting Plant Under Subcritical Conditions

Under subcritical conditions, we studied the chemical extraction of arsenic (As) from contaminated soils that were sampled from the vicinity of abandoned mines and a smelting plant in South Korea. The total initial concentrations of As in the soil samples from the Myungbong and Cheongyang mines and the Janghang smelting plant were 298.6, 145.6, and 103.7 mg/kg, respectively. X-ray photoelectron spectroscopy analysis showed that the species of As identified in the soil was As(+V), including As₂O₅ and AsO₄ ^3? _. At 20°C, only 27.4, 26.5, and 40.1% of the total As was extracted from the Myungbong, Cheongyang, and Janghang soil samples, respectively, with 100 mM of NaOH. As the temperature was increased to 300°C, the extraction efficiencies remarkably increased. However, to achieve the complete extraction of As from the soils, 100 mM of citric acid, EDTA, or NaOH was needed at 200, 250, or 300°C. Extraction with subcritical water at 300°C resulted in incomplete extraction of As from the soils. The results of these experiments indicate that extraction mechanisms other than oxidative dissolution of As(+III) species may be responsible for the enhancement of As extraction. Our results suggest that subcritical water extraction combined with extracting reagents can effectively remediate As-contaminated soil regardless of the As species.     

Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K _D was performed using a novel distillation setup allowing stripping of ethanol at 50–65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55–65 °C) and exposure to gas–liquid and liquid–heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.     

Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Absence of high levels of DNA polymerase α activity in the nuclear matrix prepared from mouse erythroleukemia cells

We have examined the association of DNA polymerase α activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase α activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37°C for 45 min prior to subfractionation. The recovery of DNA polymerase α activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH₄)₂ SO₄), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.     

Methods for DNA extraction from various soils: a comparison

Seven methods for bacterial DNA extraction and purification from soil samples were compared. Holben's direct lysis method recovered significantly greater amounts of DNA than the other methods tested, while CsCl-ethidium bromide density gradient ultracentrifugation was better than gel filtration at removing humic acid from crude DNA isolated from soil. When both these methods were combined, 5·94 μg of DNA (A_260/280 ratio around 1·754) was yielded g⁻¹ oven-dried sandstone shale alluvial soil; similarly satisfactory yields were obtained from Taiwan clay, and sandstone shale and slate alluvial soil managed under different farming practices. DNA obtained by these methods was readily digested by Eco R I and Hind III. When soil samples were stored for 3 weeks at 4 °C, the fraction of high-molecular-weight DNA was reduced significantly. Thus, DNA extraction should be carried out as soon as possible after a soil sample has been collected from the field. When hyphae of Pythium aphanidermatum and Fusarium solani were subjected to the above lysis method, DNA could not be detected in the extract.     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.