Single-cell analysis and isolation for microbiology and biotechnology: methods and applications

Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.     

RNA研究相关技术及其应用

RNA技术可以分为RNA基础研究相关的技术、RNA应用相关的技术和RNA的生物信息学技术。RNA基础研究相关技术包括RNA分离纯化和鉴定技术、RNA与其他生物大分子相互作用技术、RNA高级结构的研究技术和其他相关RNA技术;RNA应用相关技术则包括用于生产其他产品的RNA技术和直接用于药物开发的RNA技术;RNA的生物信息学技术则有各种数据库、非编码RNA的预测、RNA二级结构预测和各种设计软件。本文简略介绍了上述各类RNA技术的原理及其国内外研究进展,从而有助于对RNA领域有关技术方面有一较全面的了解。     

基因治疗载体及其基因转移技术的关键问题与研究现状

目前使用的基因治疗载体及其基因转移技术中还没有一种能用于临床并永久有效的基因转移技术 .该文分析了直接体内转移、间接体内转移及其非载体法、病毒载体法、非病毒性生物载体法等基因治疗转移技术存在的一些关键问题 ,并探讨了各问题的解决办法或研究策略以及基因治疗载体研究的发展方向     

Present and potential applications of mass spectrometry for bioprocess research and control.

For on-line monitoring of bioprocesses present applications are mainly restricted to gas analysis, but several techniques have been improved recently: membrane probes, the application of MS/MS techniques, methods of correlating available on-line data like gas reaction rates with bioprocess characteristics using stoichiometric models and other empirical correlations. New ionization and ion separation methods for biomolecules are developing dramatically. Most striking developments in this area are improved desorption techniques, electrospray, the renaissance of time-of-flight instruments and new challenges in ion trap techniques. Enormous progress is made in the analysis of peptides and other biopolymers. Combinations with new separation techniques like capillary electrophoresis and capillary HPLC show new horizons in biomolecule analysis.     

差异显示和RACE技术的发展与应用

差异显示是一套快速有效克隆差异表达基因的新技术 ,综述了该技术的原理及其技术改进、应用与展望。“快速扩增cDNA末端”(RapidAmplificationcDNAEnd)技术即RACE技术 ,是获得全长cDNA的快速有效的方法 ,阐述了该技术的发展与应用。     

昆虫抗药性监测与检测技术研究进展

综合论述了昆虫抗药性监测与检测技术的研究概况,扼要地介绍了生物检测法、神经电生理检测法、生物化学检测法、免疫学检测法和分子生物学检测法的研究进展,指出了这些检测方法存在的问题.理论的发展和试验技术的进步,为早期监测和检测昆虫抗药性提供了更多快速可行的方法,使昆虫抗药性监测与检测技术的研究迈上一个新台阶.     

第三代测序技术的研究进展与临床应用

Sanger测序法,又称第一代测序技术,作为测序金标准推动了人类基因组“工作框架图”的绘制,但通量低、成本高的缺点限制了其进一步大规模应用。第二代测序技术,又称下一代测序技术,因其通量高、成本低等优点使得基因测序在基础研究与临床诊疗中得到广泛应用,但短读长一直是其不可回避的技术短板。第三代测序技术的出现,因其具有长读长优势,为基因序列上复杂重复区域解析与高质量基因组组装提供了新的技术手段。近年来,第三代测序技术进一步发展与完善,同时在肿瘤、免疫、生殖等相关领域逐步体现出临床应用价值。本文将综述第三代测序技术的研究进展与临床应用。     

Origins of techniques in human and animal embryology

Increasingly innovative and imaginative techniques are being developed to investigate the development of animal and human embryos. Among the types of techniques that have been developed are ones that deal with oocyte maturation and culture, the isolation and utilization of stem cells, cryopreservation of reproductive cells and tissues, and various procedures to manipulate early embryos. To appreciate the derivation of these sophisticated techniques, it seems appropriate to consider the very early origins of these current techniques.     

Chromatin structure and specificity revealed by immunological techniques

The relation between the structure and function of chromatin is complex and not fully understood; therefore, a variety of experimental techniques have to be employed to elucidate the structural basis of the regulation of the genetic message encoded in DNA. The present review attempts to point out and summarize the use of antibodies as probes to study the structure and specificity of chromatin, chromosomes and their components.

The applicability of immunological techniques requires two major steps. The first step requires elucidation of a large repertoire of antibodies specific for the various forms and components of chromatin and chromosomes. The usefulness of antibodies as probes for chromatin structure is directly dependent on the number of specific, well characterized antisera. The available immunochemical techniques allow elucidation and purification of antibodies against almost any conceivable antigen. Indeed, as indicated in this review, a large number of antisera is already available. However the list of potentially useful antisera is almost unlimited. The second step involves adaptation of various aerological and immunochemical techniques to the study of chromatin and chromosomes. Unfortunately most of the techniques used are not quantitative. However, the information obtainable on both the gross and fine arrangement of individual components in chromatin and chromosomes is not easily available by other techniques.

As the techniques for preparing ‘clean’, well-defined nuclei, soluble chromatin, well-defined nucleosomes or polynucleosomes, and unfixed chromosomes improve the information obtainable by immunological techniques will be more exact and defined. Table 1 lists some uses of aerological techniques in the study of chromatin, chromosomes and their components.     

Endovascular extraction techniques for pacemaker and ICD lead extraction: Part 1

In the last few years, comprehensive endovascular techniques have been developed to extract chronically implanted pacemaker and defibrillator leads. It is important that referring physician have knowledge of the advantages and limitations of the different techniques. In this paper we discuss the techniques and results of the currently used endovascular extraction techniques.     

Relationships between xylem sap constituents and leaf conductance of well-watered and water-stressed maize across three xylem sap sampling techniques

Many different techniques have been used for xylem sap collection, but few direct comparisons of techniques have been conducted and few comparisons have been based on comprehensive analyses of xylem sap. Moreover, the suitability of extraction techniques for use on plants grown under water-stress conditions has not been addressed. Xylem sap was extracted from both well-watered and water-stressed Zea mays plants using three different techniques. The main aim was to determine how the extraction method altered the correlations between sap constituents and stomatal conductance in order to determine which relationships change with extraction technique. A 'root pressure' technique was the simplest method of extracting large volumes of sap, but the low sap delivery rates altered the composition of sap. Two pressurization techniques that varied in the position from which sap was collected were tested. The pressurization techniques allowed for the control of delivery rates that influence sap constituent concentrations. The position from which xylem sap was collected on the plant was also found to be important. All three techniques produced consistent correlations between ABA and chloride delivery rates and changes in stomatal conductance, suggesting that each technique could be applied to identify certain putative xylem-borne signals.     

蛋白质组学中的分离检测技术

10多年来,随着基因组学研究取得的巨大成就,蛋白质组学的研究也得到了突飞猛进的发展,并产生了许多先进的分离检测技术,包括与电泳相关的和非电泳的技术。本就蛋白质组学中的分离检测技术,如双向电泳、差异凝胶电泳、毛细管电泳、液相色谱质谱联用、蛋白质芯片等作一综述。     

Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract

In this review an overview of various molecular techniques and their application for the detection and identification of bifidobacteria and lactobacilli in the gastrointestinal (GI) tract is presented. The techniques include molecular typing techniques such as amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), ribotyping and community profiling techniques such as PCR coupled to temperature and denaturing gradient gel electrophoresis (PCR-TGGE and PCR-DGGE, respectively). Special attention is given to oligonucleotide probes and primers that target the ribosomal RNA (rRNA) sequences and their use in PCR and different hybridisation techniques such as DNA microarrays and fluorescent in situ hybridisation (FISH). In addition, recent findings based on the molecular studies of bifidobacteria and lactobacilli in the GI-tract are reviewed.     

Chemiluminescent and bioluminescent assays as innovative prospects for mycotoxin determination in food and feed.

Mycotoxin contamination of food and feedstuffs is among the top priorities for human and animal safety. The currently used techniques for mycotoxin determination, either chromatography or ELISA, are unsuitable for routine in-field assessment. There is an urgent need for other accurate, simple and cost-effective techniques that can be used as a screening tool for a rapid estimation of mycotoxin contamination in commodity lots. This paper reviews the literature on the use of chemiluminescence (CL) and bioluminescence (BL) assays for direct or indirect mycotoxin assessment. The chemiluminescence immunoassays, adenosine triphosphate (ATP) bioluminescence and bioassays are reviewed and their advantages and limitations discussed. These techniques used in food testing and the pharmaceutical industry offer promise as rapid techniques for mycotoxin determination. Chemiluminescence and bioluminescence bioassays are the most innovative alternatives to the conventional techniques used for mycotoxin determination in food and feed.     

生物大分子分离技术:过去、现状和未来

生物大分子包括多肽、酶、蛋白质、核酸(DNA和RNA)以及多糖等。生物大分子分离技术是生命科学研究中的关键技术之一。当前,各学科之间的交叉渗透为生物大分子分离技术的发展提供了更多的契机。对以沉淀、透析、超滤和溶剂萃取为代表的传统分离技术,以及色谱,电泳等现代分离技术的发展概况、原理、特点及应用进行了综述。并结合生命科学的发展现状,展望了生物大分子分离技术的发展前景。     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

昆虫触角电位(EAG)及其与气谱联用(GC-EAD)技术

昆虫触角电位(EAG)和气相色谱-触角电位(GC-EAD)是昆虫化学生态学中最重要的电生理技术,在研究昆虫对信息化学物质的感受机理、筛选活性信息素成分或植物次生物质等方面发挥关键作用。本文介绍这两种技术的基本原理、操作步骤和应用实例等,并对使用中应该注意的事项进行了详尽讨论。     

Advances in molecular marker techniques and their applications in plant sciences

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.     

Molecular genetics and the management and conservation of marine organisms

Biochemical and molecular species identification techniques have a broad range of applications in the management and conservation of marine organisms. While species boundaries are not always clearly defined, phylogeneticists utilise autapomorphic characters to distinguish phylogenetic species. Genetic markers discriminate between marine taxa when traditional morphological distinctions are unclear. The applications of these techniques can be divided into four general categories. Firstly, compliance enforcement, which often depends on genetic identification techniques to enable officials to identify the species to which regulations pertain. Secondly, quality control applications, to allow for the testing of marine products to guard against fraudulent substitution with less valuable species, which is particularly pertinent since processing often obliterates identifiable features. Thirdly, a variety of applications to ecological and life-history studies and conservation management are reported. Here, the genetic identification techniques of species from cryptic life-cycle stages or of morphologically indistinct species are an indispensable tool for marine scientists, conservators and managers. Lastly, the application of genetic techniques for sourcing population origin is briefly discussed. The biochemical and molecular techniques applied to species identification all exploit phenotypic or genotypic polymorphisms that are sampled using either tertiary level protein based methods or primary level DNA based methods. In this review, examples of the applications along with the total protein, allozyme, serological, PCR and other DNA based methodologies are briefly described and some generalities with regard to their use are presented.