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DGGE/TGGE技术在土壤微生物分子生态学研究中的应用 总被引:3,自引:0,他引:3
传统的微生物生态学研究方法只限于环境样品中极少部分(0.1% ̄1%)可培养的微生物类群,极大程度地限制了对土壤微生物群落结构的研究。综述了以16S rDNA为主要研究对象的DGGE/TGGE(Denaturing gradientgel electrophoresis,DGGE/Temperature gradient gel electrophoresis,TGGE)技术原理,以其为主要手段结合PCR扩增、克隆建库、序列测定以及种系分析对土壤微生物的群落结构和多样性研究的最新动态。DGGE/TGGE技术极大地推动了土壤微生物分子生态学的发展,同时也为实际问题的诊断、作物生长跟踪监测等提供了技术支撑,在土壤微生物分子生态学研究和生产实践中起着越来越重要的作用。 相似文献
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PCR-DGGE技术及其在植物微生态研究中的应用 总被引:2,自引:1,他引:1
植物中微生物种类较为丰富,在植物微生态系统中发挥重要作用.变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术作为研究微生物物种多样性和种群动态变化的分子检测工具之一,广泛应用于植物微生物群落多样性和群落动态变化研究中.概述了DGGE技术的原理,及其在植物微生物生态学中的应用以及在该领域研究中导致DGGE偏差的因素和相应解决方法,对DGGE检测结果的分析提供参考,为进一步研究植物微生物相互作用提供帮助. 相似文献
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PCR-DGGE技术在农田土壤微生物多样性研究中的应用 总被引:49,自引:6,他引:43
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。 相似文献
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这是现代生物技术在环境微生物学中的应用系列综述文章的第三篇 ,讨论限制性片段长度多态性 (RFLP)分析、变性梯度凝胶电泳 (DGGE)和温度梯度凝胶电泳 (TGGE)以及报道基因。 相似文献
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DNA指纹图谱技术在土壤微生物多样性研究中的应用 总被引:1,自引:0,他引:1
土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。 相似文献
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温度梯度凝胶电泳(TGGE)是一种用于检测核酸序列变异和点突变的电泳方法.该法利用不同构象的核酸分子具有不同的变性温度(Tm)来进行分离.TGGE方法具有分辨能力高、重复性好和节省时间的特点,可广泛应用于分子生物学研究领域. 相似文献
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DGGE/TGGE a method for identifying genes from natural ecosystems. 总被引:64,自引:0,他引:64
G Muyzer 《Current opinion in microbiology》1999,2(3):317-322
Five years after the introduction of denaturing gradient gel electrophoresis(DGGE) and temperature gradient gel electrophoresis (TGGE) in environmental microbiology these techniques are now routinely used in many microbiological laboratories worldwide as molecular tools to compare the diversity of microbial communities and to monitor population dynamics. Recent advances in these techniques have demonstrated their importance in microbial ecology. 相似文献
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Fluorophore-Labeled Primers Improve the Sensitivity, Versatility, and Normalization of Denaturing Gradient Gel Electrophoresis 总被引:3,自引:1,他引:2 下载免费PDF全文
Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology. We tested the effect of fluorophore-labeled primers on DGGE band migration, sensitivity, and normalization. The fluorophores Cy5 and Cy3 did not visibly alter DGGE fingerprints; however, 6-carboxyfluorescein retarded band migration. Fluorophore modification improved the sensitivity of DGGE fingerprint detection and facilitated normalization of samples from multiple gels by the application of intralane standards. 相似文献
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利用时间进程法优化活性污泥DG-DGGE图谱 总被引:5,自引:0,他引:5
目的:为了探讨电泳时间对双梯度-变性梯度凝胶电泳(DG-DGGE)分析活性污泥样品时的影响。方法:提取污泥DNA后,以通用引物338f/534r扩增16S rDNA序列,采用时间进程法优化PCR扩增产物的DG-DGGE分离效果。结果:采用不同电泳时间进行DGGE分析时,DGGE图谱存在显著的差异。16S rDNA V3区(200 bp)在凝胶梯度6%~12%,变性剂梯度30%~60%时,在200V电压下,最佳电泳时间为5h。 相似文献
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The composition and development of microbial population during the manufacture and ripening of two batches of a blue-veined cheese was examined by culturing and polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine selective and/or differential media were used to track the cultivable populations of total and indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences between the results obtained by the culturing and the molecular method were recorded for some populations. Culturing analysis allows minority microbial groups (coliforms, staphylococci) to be monitored, although in this study PCR-DGGE identified a population of Streptococcus thermophilus that went undetected by culturing. These results show that the characterization of the microbial populations interacting and evolving during the cheese-making process is improved by combining culturing and molecular methods. 相似文献
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Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries 总被引:1,自引:0,他引:1
Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones. 相似文献