高温环境样品总DNA直接和间接提取方法的比较

分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。     

腾冲热海四种不同颜色菌藻席的DGGE图谱比较

研究热泉水化学成分与菌藻席群落结构的关系。直接提取四种菌藻席(mat)总DNA,PER扩增获得16S rDNA的V8高变区片段,进行DGGE分析。结果：四种菌藻席的细菌组成差异很大,DGGE条带数目最低为20,最高为47;4种菌藻席共有的条带为1。结合文献报道的3个热泉的水化学性质分析,表明水化学成分是热泉生态系统中重要的生态因子,直接影响菌藻席的群落结构和物种组成。     

云南腾冲热海两热泉菌藻席细菌多样性的研究

应用显微形态观察和变性梯度凝胶电泳(DGGE)对云南腾冲热海两热泉菌藻席的细菌多样性进行了比较分析.直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,得到包含V8和V9高变区的16S rDNA片段,进行DGGE分析,结合形态观察,结果显示,热泉菌藻席中存在丰富的细菌多样性,且不同温度范围的菌藻席细菌组成差异显著.     

云南腾冲热海三热泉细菌多样性的研究*

应用变性梯度凝胶电泳 (DGGE)对云南腾冲热海 3个高温热泉中菌藻席和泉底沉积物的细菌多样性进行了初步研究。直接从环境样品中提取总DNA ,用两套细菌通用引物进行PCR扩增 ,分别得到包含V₈和V₉高变区的 1 6SrDNA片段 ,进行DGGE分析。结果表明 :菌藻席和泉底沉积物中不仅物种组成差异较大 ,而且都存在丰富的细菌多样性 ;一些关键的生态因子 ,如氧气、温度等会对群落中微生物的物种组成有很大影响。     

云南腾冲热海三热泉细菌多样性的研究

应用变性梯度凝胶电泳(DGGE)对云南腾冲热海3个高温热泉中菌藻席和泉底沉积物的细菌多样性进行了初步研究。直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,分别得到包含V8和V9高变区的16SrDNA片段,进行DGGE分析。结果表明：菌藻席和泉底沉积物中不仅物种组成差异较大,而且都存在丰富的细菌多样性;一些关键的生态因子,如氧气、温度等会对群落中微生物的物种组成有很大影响。     

工业化废水处理反应器污泥总DNA提取方法

根据工业化废水处理反应器污泥特性,对常规的溶菌酶-SDS-酚/氯仿环境样品总DNA提取方法进行改进,增强样品预处理,强化细胞裂解,提高杂质去除效率,获得了一种工业化污泥总DNA提取的通用方法,并采用该方法对石家庄若干实际运行的工业化厌氧、好氧反应器的污泥样品进行了总DNA提取研究.结果表明,该方法对所选污泥样品均有效,具有普适性.提取的污泥总DNA杂质含量少,纯度高,A260/A280在1.8左右;提取效率较高.总DNA产率都在0.7 mg/g以上,最大产率可达0.85 mg/g.所提取的污泥总DNA可以直接作为模板进行PCR反应,PCR产物直接进行变性梯度凝胶电泳(DGGE),能够得到较好的DGGE谱图,表明该方法提取的污泥总DNA样品可满足后续分析研究的要求.     

三种粪便总DNA提取方法的比较

目的比较不同粪便总DNA提取方法对肠道菌群多样性研究的影响。方法采用Bead beating法、化学裂解法和QIAamp DNA Stool Mini Kit提取同一份人粪便样品的总DNA,对比3种方法的DNA得率和16S rRNA基因V3区的变性梯度凝胶电泳（DGGE）图谱。结果Bead beating法的DNA得率约是其他2种方法的2倍;3种方法得到的DGGE图谱的Dice相似性为60％～70％,2条优势条带只出现在Bead beating法图谱中。在2～5min的Bead beating法击打时间里,DNA得率随击打时间的延长有一定的增加,但DGGE图谱无显著变化。结论不同的DNA提取方法会影响菌群的多样性分析。比较其他2种方法,Bead beating的裂解效率更高,能够检测到更多种类的细菌,更合适肠道菌群组成的分子研究。     

堆肥中微生物总DNA的高效提取

采用化学裂解和酶解相结合的方法,选择加入PVPP的高盐缓冲液作为细胞裂解的反应体系,并以PEG-8000进行DNA沉淀,从高有机含量的堆肥样品中进行微生物总DNA的提取。结果表明,从4种性质不同的堆肥中均获得了高质量的微生物总DNA,所得的DNA分子片段在23kb左右;每克干重堆肥的总DNA提取量为63.54±12.08μg~106.50±28.36μg,A260/A280大于1.6,A260/A230大于1.8,不用经过纯化可以直接进行PCR扩增和限制性酶切;以该DNA为模板进行微生物区系的DGGE分析,显示了丰富的微生物多样性。该方法减少了通常环境样品DNA提取过程中的纯化步骤,减少了DNA的损失,为从事微生物分子生态学,尤其是那些针对高有机含量以及获取极为不易的环境样品的研究而言是十分有益的。     

基于PCR DGGE的小鼠肠道菌群基因组提取方法的建立

通过比较4种小鼠粪便细菌总DNA提取方法对基于PCR-DGGE检测的肠道菌群多样性分析的影响,旨在建立适于PCR—DGGE的小鼠肠道微生物宏基因组提取的稳定、经济、快捷的方法。采用SDS裂解法、某国产市售粪便DNA提取试剂盒、改进的化学裂解法、改进的溶菌酶法4种方法提取小鼠粪便细菌总DNA,通过琼脂糖凝胶电泳、紫外分光光度法、细菌16S rRNAV3区PCR扩增结合DGGE对提取结果进行比较分析。SDS裂解法和国产市售试剂盒2种方法提取粪便细菌总DNA均未得到理想结果,另2种方法均能够检测到粪便中20种左右的细菌。改进的化学裂解法和改进的溶菌酶提取法的建立为基于PCR—DGGE进行肠道菌群结构的定量及定性分析提供了可靠的前提基础和实验保障。     

几种直接从高温热泉沉积物中提取DNA方法之比较

用酶解法,化学裂解法和微波炉法,冻融法等物理裂解方法组合出5种方法直接从滇西一个高温热泉沉积物提取DNA。经常规的琼脂糖凝胶电泳检测,发现用溶菌酶,蛋白酶K酶解,SDS化学裂解结合微波炉法裂解不仅可以得到较大量的DNA。而且碎片段较少,提取出来的DNA经RNA酶和蛋白酶K处理后可直接进行PCR扩增。同时DGGE（变性梯度凝胶电泳）电泳检测,结果表明,此法不仅可以得到该种环境中较多微生物分类单位的DNA,而且还能够较好地体现出它们在量上的差异。     

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh,India to Explore Bacterial Diversity

Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.     

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.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Differential microbial communities in hot spring mats from Western Thailand

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.     

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.