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1.
Kurt. E. Williamson Jinjun Kan Shannon J. Williamson 《Soil biology & biochemistry》2011,43(4):736-748
The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population. 相似文献
2.
土壤宏基因组学研究方法与进展 总被引：3，自引：0，他引：3
土壤微生物驱动着土壤中的物质循环和养分转化。在土壤学的研究中,长期将土壤作为一个黑箱系统来对待,对其中的生物组成及其参与的生化过程知之甚少。土壤中绝大部分微生物目前尚难以分离培养,因此基于传统的培养方法对于认识土壤微生物群落组成和功能有其局限性。宏基因组学直接从环境样品中提取全部微生物的DNA,或通过测序探究环境中微生物的群落结构和功能(序列驱动),或构建宏基因组文库,筛选新的基因或生物活性物质(功能驱动),克服了传统培养方法的缺陷,极大地丰富了对土壤微生物多样性及其功能的认知。本文在综述土壤宏基因组学研究基本流程的基础上,重点介绍了日益重要的第二代测序平台在土壤宏基因组学研究中的应用及其产生的海量数据的分析处理方法,并简要探讨了宏基因组学在土壤微生物生态学中的应用。最后,作者建议在国家层面上展开相关土壤宏基因组学研究,调查微生物群落及其变化,为生物资源开发、农业生产和环境保护作出应有的贡献。 相似文献
3.
Extraction of DNA from soil 总被引：1，自引：0，他引：1
Patrick Robe Renaud Nalin Carmela Capellano Timothy M. Vogel Pascal Simonet 《European Journal of Soil Biology》2003,39(4):183-190
There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purification. The alternative approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purification. Several published methods describe the recovery of highly purified nucleic acids that are well-suited for molecular purposes even though a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective. 相似文献
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传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一 相似文献
6.
从水牛瘤胃内容物的添加滤纸为碳源的富集培养物中提取未培养微生物的总DNA,以柯斯质粒为载体构建了1个含约8000个克隆的宏基因组文库,对文库进行活性筛选获得1个既表达CMCase活性又表达4-MUCase酶活性的克隆。亚克隆及测序分析发现1个潜在的可编码333个氨基酸的ORF(Open Reading Frame),其蛋白质产物与1个来源于未培养细菌的糖苷水解酶家族5的纤维素酶Cel A的同源性最高,两者的一致性为53%,相似性为68%。将PCR扩增的该基因完整的ORF克隆入表达载体pET30a(+),在大肠杆菌中得到其过量表达产物。经过Ni-NTA纯化后,该表达产物(Umcel5K)具有CMCase活性和4-MUCase酶活性,其最适pH是4.5~5.0,最适温度是50°C。pH耐受性检测表明,该酶在pH4~4.5比较稳定。温度耐受性实验表明该酶不耐高温,在55°C以下比较稳定。经过镍柱纯化的酶液比活为26.15 U/mg。部分金属离子如Fe3+、Cr2+或Cu2+会抑制该酶的酶活,而另外一些金属离子如K+、Li+等对Umcel5K的活性影响不大。 相似文献
7.
Following the need for methods suitable for the assessment of the habitat function of soil (i.e. the ability of soil to act as an environment for organisms), particularly in Europe, the International Organization for Standardization (ISO) is harmonizing a standard called “soil quality—sampling of soil invertebrates” now include as many as four parts that are currently close to finalization: (1) ISO/DIS 23611-1: hand-sorting and formalin extraction of earthworms; (2) ISO/DIS 23611-2: extraction of microarthropods (Collembola and Acarina); (3) ISO/CD 23611-3: extraction of enchytraeids; (4) ISO/WD 23611-4: sampling, extraction and identification of free-living stages of terrestrial nematodes All drafts cover the technical details of the most appropriate methods, but also contain modifications of the methods required in special cases (e.g. when working in different climatic regions like the tropics). It is proposed to use these methods in all studies collecting data on soil organism communities for legal purposes such as long-term monitoring or soil quality assessment. 相似文献
8.
Ribosomal intergenic spacer analysis (RISA) has been applied to the microbial community analysis of agronomic products in combination with a simple and rapid DNA extraction method, consisting of a one-step extraction and two-step purification, for a variety of agronomic products. RISA appears to be a useful tool for the study of the community structures of food-associated microbes and their use as a unique fingerprinting signature for each agronomic product. Sequencing analyses of amplicons generated from RISA suggest that this method can detect conventional microbes. In the case of RISA of wasabi paste DNA, the sequences of the amplicons showed high similarity to the plant pathogen Xanthomonas campestris and the soil bacterium Bacillus subtilis, whereas several food-associated bacteria (Lactococcus lactis, Lactococcus raffinolactis, and Lactococcus sakei) were detected using this technique in sausage DNA. Unexpectedly, the sequencing analyses also revealed the presence of several microbes that possessed high similarity to human bacterial pathogens such as Weissella confusa and Yersinia pestis. The results suggest that RISA will be a useful method for routine microbial community analysis in agronomic products. 相似文献
9.
Soil faunal communities are often phylogenetically diverse and the accurate assessment of the taxonomic structure of these communities is both time-consuming and requires a high level of taxonomic expertise. Here we describe a DNA sequence-based methodology for characterizing soil micro- and mesofaunal communities that is similar to the molecular approaches commonly used to survey soil microbial diversity. The technique involves the direct extraction of faunal DNA from soil, PCR amplification of the extracted DNA with metazoan-specific primers, followed by the construction of clone libraries and direct sequencing of individual PCR products. We used this technique to characterize micro- and mesofaunal community composition from six individual soils representing two land-use types. The technique captured the more abundant faunal groups in the soils (nematodes, Collembola, Acari, tardigrades, enchytraeids) and provided sufficient taxonomic resolution to describe the overall structure of the communities. We compared the results obtained using this molecular approach to results obtained using a traditional, microscopy-based approach and found that the results were broadly similar. However, since biases are inherent in both methods it remains unclear which method provides a more accurate assessment of soil faunal community composition. Although this molecular approach has some distinct disadvantages over the more widely-used direct extraction methods, one advantage is that the taxonomic identification it can provide will be more accurate and consistent across research groups, facilitating effective comparisons of mesofaunal surveys. 相似文献
10.
猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。 相似文献
11.
Background, aim and scope An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires
extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a
larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still
have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR
inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids
can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg2+ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects
of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were
assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine
plantation of southeast Queensland, Australia.
Materials and methods Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash
pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega
Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to
purify the DNA for PCR amplification, which include PVPP, Sephadex TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification
methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined
by successful PCR amplifications.
Results and discussion The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour
and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions
had been made from neat to 1:103. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration
decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA
losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™
spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and
soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification,
but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in
this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may
be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and
also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification.
Conclusions A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first
introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method
for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification
inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap,
fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely
used to relieve the heavy working load of molecular-biological researchers.
Recommendations and perspectives This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is
effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified
gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect
of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples
which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples. 相似文献
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A. S. Cherobaeva A. L. Stepanov I. K. Kravchenko 《Moscow University Soil Science Bulletin》2011,66(2):50-57
A comparative analysis of five methods of extraction and purification of soil DNA, including a modification of the authors,
was performed for the further molecular investigation of various ammonium-oxidizing bacteria and archaea in soils. Experiments
using soil samples from natural ecosystems and agroecosystems of the European area of Russia established that the amount of
DNA extracted by different methods depended significantly on the type of soil. The subsequent molecular analysis (PCR-DGGE)
of ribosomal (16S rRNA) and functional (amoA) genes demonstrated significant differences in the community structure of ammonium oxidizers depending on the method of DNA
extraction. The best results were obtained for acidic soil (soddy-podzolic and gray forest soils) when using the method of
Griffiths et al. [4] with our own modification. On the other hand, application of commercial DNA extraction kits was most
efficient for soils with a high content of humus (black and chestnut soils). According to the results obtained, molecular
analysis of soil microbe communities required selection of optimum conditions for DNA extraction, especially for soils with
high contents of organic compounds and clay minerals at different pH levels. 相似文献
14.
由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50％以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3／pthA家族基因的情况,本研究应用Alu I部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHI酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27．7kb,最大为58．5kb,平均大小为39．9kb,文库克隆容量约为2．8×10^3Mb,该文库中包含基因组中任一个基因的概率为99．4％。利用来自水稻白叶枯病菌菲律宾菌株PX086的无毒基因avrXa10的第252位～第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3／pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3／pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3／pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3／pthA家族基因的功能奠定了基础。 相似文献
15.
Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis 总被引：1，自引：0，他引：1
Julia R. de Lipthay Christiane Enzinger Kaare Johnsen Jens Aamand 《Soil biology & biochemistry》2004,36(10):1607-1614
The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding-freezing-thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel electrophoresis. This indicates the importance of the DNA extraction protocol on data for evaluating soil bacterial diversity. Consistently, the bead-beating procedure gave rise to the highest number of DNA bands, indicating the highest number of bacterial species. Supplementing the bead-beating procedure with additional cell-rupture steps generally did not change the bacterial community profile. The same consistency was not observed when evaluating the efficiency of the different methods on soil DNA yield. This parameter depended on soil type. The DNA size was of highest molecular weight with the sonication and grinding-freezing-thawing procedures (approx. 20 kb). In contrast, the inclusion of bead beating resulted in more sheared DNA (approx. 6-20 kb), and the longer the bead-beating time, the higher the fraction of low-molecular weight DNA. Clearly, the choice of DNA extraction protocol depends on soil type. We found, however, that for the analysis of indigenous soil bacterial communities the bead-beating procedure was appropriate because it is fast, reproducible, and gives very pure DNA of relatively high molecular weight. And very importantly, with this protocol the highest soil bacterial diversity was obtained. We believe that the choice of DNA extraction protocol will influence not only the determined phylogenetic diversity of indigenous microbial communities, but also the obtained functional diversity. This means that the detected presence of a functional gene—and thus the indication of enzyme activity—may depend on the nature of the applied DNA extraction procedure. 相似文献
16.
为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法. 相似文献
17.
Zhanbei Liang Rhae A. Drijber Donald J. Lee Ismail M. Dwiekat Steven D. Harris David A. Wedin 《Soil biology & biochemistry》2008,40(4):956-966
Although arbuscular mycorrhizal fungi (AMF) are crucial for ecosystem functioning, characterizing AMF community structure in soil is challenging. In this study, nested polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were combined with cloning of fungal 18S ribosomal gene fragments for the rapid comparison of AMF community structure in soil. Reference AMF isolates, representing four major genera of AMF, were used to develop the method. Sequential amplification of 18S rDNA fragments by nested PCR using primer pairs AM1-NS31 and Glo1-NS31GC followed by DGGE analysis yielded a high-resolution band profile. In parallel, 18S rDNA fragment clone libraries were constructed and clones screened by DGGE. Sequence identity was inferred by matching the electrophoretic mobility of the sample fingerprint bands to that of bands from individual clones. The effectiveness of this approach was tested on soil samples from different ecosystems, yielding reproducible, complex DGGE band patterns specific to each site. The coupling of PCR–DGGE with clone library analysis provides a robust, reliable, and precise means to characterize AMF community structure in soils. 相似文献
18.
In criminal investigations, information on the origin of soils may be crucial for solving cases. The biological complexity of soil may potentially be used for sorting and differentiating between soil samples. Nucleic-acid based analyses of soil microbial populations are powerful tools, routinely used in studies of this habitat. Application of such approaches in forensics implies that a standardized DNA extraction method has to be applied to all samples. In this study, several DNA extraction protocols were compared. An improvement on the method proposed by Tsai and Olson (1991) was found to be most suited to extract DNA from various soil types, including from small samples. A blind test on soils from a crime, an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and denaturating gradient gel electrophoresis for use in forensic science. In most cases, soil patterns clustered according to soil type and location. 相似文献
19.
A pre-lysis buffer washing procedure was introduced to DNA extraction from a forest soil with high organic matter and iron oxide contents. Sodium phosphate of 0.1 M (pH 7.5) was used as a buffer to wash soil samples when subsequent lysis buffer was phosphate, and 20 mM EDTA (pH 7.5) was used when subsequent lysis buffer included EDTA. Initial experiments were not successful because the DNA extracts could not be amplified by polymerase chain reaction (PCR). The consideration of introducing a pre-lysis washing procedure was based on the idea that the washing should promote soil dispersion and homogeneity, decrease DNA adsorption by soil components (e.g. iron oxides), and remove covalent cations and those easily-dissolving organic compounds from the soil samples. Results revealed that humic substance content decreased by 31%, but DNA yield increased by 24% in the DNA extracts of the pre-lysis washing procedures, compared to the non-washing procedures. DNA extracted by the pre-washing procedure needed less purification for subsequent 18S and 16S rDNA PCR amplifications. It was recommended that the pre-lysis buffer washing should be used for DNA extraction from those difficult environmental samples, such as the forest soil with high contents of organic matter and iron oxides. 相似文献
20.
传统的微生物分离培养方法,在反映茶园土壤微生物基因信息上有很大的局限性,因此,目前逐步被分子生态学的方法替代,而获得高质量、大片段、无偏好的土壤微生物总DNA则是茶园土壤微生物分子生态学研究的基础。本文采用SDS高盐法、变性剂加SDS高盐法、脱腐SDS高盐法、CTAB法和Krsek改进法5种土壤微生物DNA提取方法分别从茶园土壤微生物中提取总DNA,并对5种方法提取的DNA的片段大小、质量和产量进行了综合评价。结果表明,Krsek改进法提取到的DNA片段最大（〉23kb）、纯度最高（OD2UOD280〉1．70;OD2UOD230〉1．35）、产量较高（〉34．50μg／gdrysoil）且不需纯化就可以用于PCR扩增和RFLP分析。因此,Krsek改进法是一种高效、可靠且适合于茶园土壤微生物分子生态学研究的DNA提取方法。 相似文献