紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

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.     

猪粪和土壤样品中微生物DNA提取方法的比较

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。     

土壤微生物总DNA提取方法的比较

采用4种土壤DNA提取方法提取了5种类型土壤的微生物总DNA，并对4种提取方法的DNA提取效果进行综合分析，进一步通过PCR—DGGE扩增提取DNA中的细菌16S rDNA片段，并对扩增产物进行DGGE电泳分析。结果表明，SDS-高盐缓冲液法抽提的DNA得率最高，SDS-酚氯仿抽提法的DNA得率最低。DNA的纯度，以BIO-101 DNA Extraction Kit试剂盒法最高，改进试剂盒法纯度最低。通过PCR-DGGE分析土壤微生物多样性结果表明，BIO-101 DNA Extraction Kit试剂盒法提取的DNA代表的微生物多样性最全，而SDS酚氯仿法抽提的DNA代表性最差。     

猪粪和土壤样品中微生物DNA提取方法的比较

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。     

茶园土壤微生物总DNA不同提取方法的比较

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。     

黄瓜白粉菌基因组DNA 提取方法比较

以黄瓜白粉病菌为试验材料,比较分析了改良CTAB法、SDS法和真菌试剂盒法对黄瓜白粉病菌基因组DNA提取的效果。结果表明,CTAB法提取的黄瓜白粉菌基因组DNA在纯度(R=A260 nm /A 280 nm)和产量上均优于SDS法和真菌试剂盒法,且杂质少。CTAB法提取的黄瓜白粉菌基因组DNA产率为204.3 μg/g,而SDS法和真菌试剂盒法分别为147.7、117.7 μg/g,且方法产率之间差异极显著。采用CTAB法提取的DNA纯度较高,为1.969 6;SDS法和真菌试剂盒法提取的DND纯度较低,分别为1.832 2和1.507 9。     

RAPD分析用白花丹参叶片DNA的提取方法

本研究采用改良CTAB法和SDS法提取新鲜白花丹参叶片的基因组DNA,初步筛选RAPD扩增引物。结果表明改良CTAB法提取的DNA较SDS法质量好,随机引物p2扩增条带相对较为清晰。再利用p2随机引物对2种方法提取的DNA进行RAPD检测比较,结果显示仅改良CTAB法能扩增出有效条带,说明改良CTAB法更适合用于白花丹参基因组DNA的RAPD检测分析。本文将为白花丹参叶片DNA的提取提供方法学参考。     

干制羊肉基因组DNA不同提取方法的比较

为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL^-1,A₂₆₀/A₂₈₀值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。     

异养氨氧化细菌基因组DNA的检测方法比较

氨氧化细菌是参与土壤氮素循环的重要微生物类群之一,其基因组DNA提取质量的准确分析,可直接影响后续分子实验的可行性和精确性。本试验针对3株异养氨氧化细菌的纯培养菌株,应用琼脂糖凝胶电泳、微量紫外分光光度计和Qubit荧光计分别检测不同提取方法获得的基因组DNA的浓度,同时结合细菌通用引物扩增16S r DNA全长来判定提取DNA的质量,进而筛选出可用于检测可培养氨氧化细菌基因组DNA浓度的方法。研究结果表明,针对不同浓度的DNA样品,尽管3种检测方法获得的结果表现出明显差异,但在16S r DNA-PCR中均仍能获得良好的扩增结果。与微量紫外分光光度法相比,Qubit方法对基因组DNA浓度的检测结果更为精确,特别在低浓度DNA检测中,能够较真实的反映基因组DNA的实际情况。     

土壤微生物多样性研究的新方法

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     

三种土壤微生物总DNA提取方法的比较

本文对3种常用的土壤微生物总DNA提取方法Martin法、高盐改进法及试剂盒法进行了比较,并通过DNA得率、纯度及16S rDNA V3可变区的PCR扩增结合DGGE法（denatumg gradient gel electrophoresis）,分别对3种方法进行评价。结果表明,3种方法提取的DNA均能满足土壤微生物多样性分析的要求。其中试剂盒方法操作简单,提取的DNA质量较高,但DNA得率较低且成本昂贵。Martin法和高盐改进法用时较长,DNA得率较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。     

Importance of DNA quality in comparative soil microbial community structure analyses

This study assessed how different in-situ lysis soil DNA extraction methods influence the DNA yield, quality and hence the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA). Of the methods tested in three soils, a modified hexadecyltrimethylammonium bromide-dithiotreitol (CTAB-DTT)-based method produced ?3 times more DNA of higher quality than the other methods (260/230 nm ratios=1.64–1.82 and 260/280 nm ratios=1.82–1.89 and extracts were less inhibitory of PCR). DNA extracted by this method also yielded more reproducible ARISA ribotypes (89?119 for bacteria and 48?88 for fungi; P<0.05) than DNA extracted by other methods, and consequently produced more reliable estimates of bacterial and fungal diversity in all three test soils. The significant correlations observed between the numbers of reproducible ribotypes and DNA extract 260/230 nm ratios (r=0.88 and 0.72 for bacteria and fungi, respectively; P<0.001) reaffirmed the strong influence of DNA quality on the reliability of microbial diversity indices determined based on PCR-based DNA fingerprinting technique. Results of discriminant function analysis (DFA) and multivariate analysis of variances (MANOVA) performed on ARISA profiles (number and relative abundance of ribotype) revealed that the variability associated with DNA extraction methods did not exceed the biological variability among soil types; this supports the conclusion that high-quality DNA underpins DNA fingerprinting techniques.     

Comparison of several methods for the extraction of DNA from potatoes and potato-derived products

Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods.     

Optimizing the indirect extraction of prokaryotic DNA from soils

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.     

短序十大功劳基因组总DNA提取方法研究

以短序大功劳嫩叶为材料,采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率,用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明,五种方法均能从短序大功劳叶片中提取到基因组DNA,但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高,可直接用于下游分子生物学实验,CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差,不利于下游的分子生物学实验;五种方法提取的总DNA的得率在10．836-451．709μg/g之间,呈CTAB法〉SDS法〉CTAB改良法1〉CTAB改良法2〉试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。     

Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

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.     

Comparative analysis of soil DNA extraction methods to study various ammonium-oxidizing bacteria and archaea by PCR-DGGE

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.