Methods for DNA extraction from Candida albicans

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.     

A comparison of methods for total community DNA preservation and extraction from various thermal environments

The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments.     

直接从土壤中提取DNA的方法

研究微生物的多样性 ,即微生物的种类和数量的多少是评价土壤质量的重要指标。由于土壤微生物种类繁多 ,数量巨大 ,加上土壤中 99%的种类难以通过传统的平板分离技术来进行培养[1],人们必须借助其他技术来解决。近年发展的非培养技术 ,如ＢＩＯＬＯＧ微量板分析技术[2 ]、细胞壁磷脂酸分析技术[3]和分子生物学方法[4 - 6 ],克服了培养的环节 ,对微生物生态学研究产生极大的推动作用。其中分子生物学是应用最广、最有发展潜力的技术。它的主要步骤是通过直接提取土壤中的ＤＮＡ ,经纯化处理后 ,利用合适的引物扩增 1 6ＳｒＲＮＡ基因 ,通过分…     

DNA extraction from soils: old bias for new microbial diversity analysis methods

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.     

Method for rapid DNA extraction from bacterial communities of different soils

A method for indirect DNA extraction from various soils significantly differing in their physicochemical properties has been developed. The proposed method is based on cell desorption from soil particles using a Tris-EDTA (TE) buffer supplemented with polyvinylpolypyrrolydone (PVPP) and sodium dodecylsulfate (SDS). Subsequent cell lysis and purification of DNA preparations methods based on alkaline lysis followed by chromatography on ion-exchange resins were described by us earlier. The purity of the DNA preparations obtained did not depend on the type of soil. It was shown that the DNA preparations can be used for the amplification of rather large fragments, e.g., sequences spanning the complete 16S rRNA gene.     

Methods for microbial DNA extraction from soil for PCR amplification

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.     

Method for rapid DNA extraction from bacterial communities of different soils

A method for indirect DNA extraction from various soils significantly differing in their physicochemical properties has been developed. The proposed method is based on cell desorption from soil particles using a Tris-EDTA (TE) buffer supplemented with polyvinylpolypyrrolydone (PVPP) and sodium dodecylsulfate (SDS). Methods for subsequent cell lysis and purification of DNA preparations based on alkaline lysis followed by chromatography on ion-exchange resins were described by us earlier. The purity of the DNA preparations obtained did not depend on the type of soil. It was shown that the DNA preparations can be used for the amplification of rather large fragments, e.g., sequences spanning the complete 16S rRNA gene.     

A rapid DNA extraction method for PCR amplification from wetland soils

Aims: We tested a method of rapid DNA extraction from wetland soil samples for use in the polymerase chain reaction. Methods and Results: The glass bead/calcium chloride/SDS method obtained in the present study was compared with the calcium chloride/SDS/enzymatic extraction method and the UltraClean? Soil DNA Isolation Kit. Rapid DNA extraction could be completed within about two hours without purification steps. Conclusions: This study succeeded in establishing a fast soil DNA extraction protocol that can be applied to various environmental sources that are rich in humic acid content. Significance and Impact of the Study: The method provides a technology with high‐quality DNA extraction from soils for testing the diversity of AOB and AOA.     

Direct extraction of DNA from soils for studies in microbial ecology

Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.     

Direct extraction of microbial community DNA from humified upland soils

This paper describes a protocol effective at extracting high yields of high-purity microbial community DNA from humified soils. DNA was extracted from soil by lysozyme, SDS and freeze–thaw lysis, precipitated and then subjected to a double caesium chloride density gradient centrifugation stage before concentrating and washing. Evaluation using three soils yielded up to 30 μg DNA g⁻¹ dry soil, with absorbance ratios at 260 : 230 nm and 260 : 280 nm of 1·6–2·0. The DNA extracted from the three soils was digested by four restriction enzymes and a 16S rDNA eubacterial product was amplified by PCR. These tests indicated that the DNA obtained by the protocol was sufficiently pure for molecular biological analysis.     

Rapid extraction of DNA from diverse soils by guanidine thiocyanate method

Molecular methods are being frequently used for the study of soil microbial communities as majority of naturally occurring microbial populations are non-culturable. In the present study, we describe a protocol of DNA extraction from diverse soils using a combination of heat, enzyme (lysozyme) and guanidine thiocyanate. The efficacy of the procedure was evaluated in terms of yield, purity and duration of extraction. The protocol was effective for neutral, acidic as well as alkaline soils (pH range 4.5-8.5). The extracted soil DNA was observed with negligible shearing on agarose gel and the time taken for restriction digestion was very less. Further, the DNA extracted was almost completely devoid of contaminants and pure enough which could be used for PCR amplification and Southern hybridization.     

DNA extraction method affects microbial community profiles from soils and sediment

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

提取北方土壤真菌DNA的一种方法

由于土壤理化性质的复杂性和真菌细胞壁结构的特殊性,从土壤样品中提取真菌基因组DNA比较困难.中国北方土壤与其它地区土壤相比有其自身的特点,因此,有必要优化一种适合于北方土壤真菌DNA提取的方法.本实验向灭菌黑土中分别投加12种在系统分类上差别较大的真菌,以传统土壤总DNA提取方法及纯菌DNA提取方法为基础,分别与蜗牛酶,纤维素酶进行组合、优化,得到7种不同的土壤真菌基因组DNA提取方法.利用真菌28S rDNA通用引物U1/U2-GC PCR-DGGE分析方法分别考察了7种不同方法所提取土壤真菌基因组DNA的多样性和代表性.结果表明:1)液氮研磨,纤维素酶、蜗牛酶和溶菌酶(浓度分别为6 、3和1 mg·ml-1)37 ℃作用60 min,2% SDS于65 ℃裂解30 min;2)-65 ℃～65 ℃冻融3次,纤维素酶、蜗牛酶和溶菌酶(浓度分别为6、3和1 mg·ml-1)37 ℃作用180 min,2%SDS于65 ℃裂解30 min的组合具有较好的提取效果.利用后一种方法分别对3种理化性质差异较大的中国北方自然土壤样品真菌DNA进行提取并分析,表明所提取土壤基因组DNA真菌特异性PCR-DGGE图谱条带丰富,该方法可用于多种北方土壤真菌多样性研究.     

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.     

Quantification of bias related to the extraction of DNA directly from soils

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.     

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.     

微量植物样本的DNA提取方法研究

为建立一种适于法庭科学实践的植物物证DNA提取优化方法,以期获得高质量的适于PCR分析的模板DNA.用8种方法从不同植物的干叶片中提取DNA,利用线粒体DNA非编码区的PCR扩增结果分析评价提取DNA的质量.结果表明8种DNA提取方法所提取的DNA都可以获得线粒体DNA非编码区的PCR扩增产物,对照紫外波长扫描结果显示,以改进的CTAB方法制备的模板DNA纯度最高,可达到进口试剂盒同等制备精度,OD260/280稳定在1.7～1.9之间.因此改进的CTAB方法适用于微量植物样本的DNA提取,可应用于法庭科学实践.     

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.     

Methods of energy extraction from microalgal biomass: a review

The potential of algal biomass as a source of liquid and gaseous biofuels is a highly topical theme, The process operations for algal biofuel production can be grouped into three areas: growth, harvesting and energy extraction, with a wide range of combinations of unit operations that can form a microalgal biofuel production system, but as yet there is no successful economically viable commercial system producing biofuel. This suggests that there are major technical and engineering difficulties to be resolved before economic algal biofuel production can be achieved. This article briefly reviews the methods by which useful energy may be extracted from microalgae biomass: (a) direct combustion, (b) pyrolysis, (c) gasification, (d) liquefaction, (e) hydrogen production by biochemical processes in certain algae, (f) fuel cells, (g) fermentation to bioethanol, (h) trans-esterification to biodiesel, (i) anaerobic digestion.