DNA分子标记技术及其在药用植物研究上的应用前景

ＤＮＡ分子标记技术及其在药用植物研究上的应用前景傅荣昭１邵鹏柱２高文远３孙勇如１１．中国科学院遗传研究所,北京１００１０１２．香港中文大学生化系香港·新界·沙田３．中国医学科学院、中国协和医科大学药用植物研究所北京１０００９４ＤＮＡ分子标记（ＤＮＡｍｏｌｅｃｕｌａｒｍａｒｋｅｒｓ）或称遗传标记（ｇｅｎｅｔｉｃｍａｒｋｅｒｓ）本质上是指能反映生物个体或种群间基因组中某种差异特征的ＤＮＡ片段。这...     

基于酿酒酵母的大片段DNA组装与转移技术进展*

DNA组装与转移技术是合成生物学的核心使能技术之一,生命体设计改造的复杂度不断提升,使得对大片段DNA组装与转移技术的需求也日益旺盛。小片段DNA的组装与转移技术目前已经比较成熟,大片段DNA由于其分子量大、易断裂,使得体外操作繁琐且效率低下。聚焦酿酒酵母体内组装和转移的技术进展,详细介绍了基于酿酒酵母一次组装和迭代组装的不同方法,并从导入与导出的角度介绍了大片段DNA的转移技术,便于研究者更好地理解和选择酿酒酵母体内组装与转移技术。此外,还展望了将酿酒酵母开发为大片段DNA组装与转移通用平台实现更多物种基因组大尺度设计改造的愿景。     

A high-throughput protocol for extracting high-purity genomic DNA from plants and animals

DNA extraction techniques that employ the reversible binding of DNA to silica via chaotropic salts can deliver high-quality genomic DNA from plant and animal tissues, while avoiding the use of toxic organic solvents. Existing techniques that use this method are either prohibitively expensive, or are applicable to only a restricted set of taxa. Here we describe a cost-effective DNA extraction technique suitable for a wide range of plant and animal taxa that yields microgram quantities of high-molecular-weight genomic DNA at a throughput of 192 samples per day. Our technique is particularly robust for tissue samples that are insoluble or are rapidly discoloured or oxidized in standard DNA extraction buffers. We demonstrate the quality of DNA extracted using this method by applying the amplified fragment length polymorphism technique to plant species.     

Site-specific analysis of drug interactions and damage in DNA using sequencing techniques

DNA-sequencing techniques can be adapted to provide powerful analytical tools for pinpointing the sites at which DNA is modified by either radiations or chemicals. Base modifications, covalent adducts, crosslinks, and noncovalent binding can all be detected. This review outlines the adaptions of the Maxam-Gilbert and Sanger dideoxy sequencing techniques which make such studies possible. Practical aspects and limitations of the various methods are given. Assays which test the ability of DNA polymerase to bypass damage and misincorporate bases are also discussed. It is concluded that these techniques constitute the most powerful method currently available for the study of sequence-specific DNA interactions.     

Comparison of the structures of operator DNA free and in complex with lambda repressor

The structures of operator DNA unbound and in complex with lambda repressor protein are compared. The conformation of the left 10 base pairs of a lambda right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to lambda repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of lambda repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.     

DNA profiling

Although some concerns still remain in standard DNA profiling technology over the assumptions from population genetics used to calculate expected match frequencies, forensic scientists are preparing for the introduction of the next generation of DNA profiling techniques based on the polymerase chain reaction. These new techniques offer the prospect of dramatically increasing the speed and sensitivity of DNA profiling and have already been applied in some casework studies.     

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.     

Hybridization histochemistry or in situ hybridization in the study of neuronal gene expression

1. Currently popular techniques of in situ hybridization histochemistry for the detection of cellular nucleic acids (DNA or RNA) are reviewed. 2. The advantages of single stranded DNA or RNA probes are discussed, together with the advantages of radioactive versus non-radioactive detection of nucleic acid signal. 3. Improving techniques of non-radioactive labelling and the use of image analysis for quantitation of radioactive signals will greatly expand the use of in situ techniques which will become commonplace in the laboratory.     

Molecular epidemiology for the detection of outbreaks of nosocomial aspergillosis

The most important molecular techniques used in the diagnosis of Aspergillus spp. and typing of the most pathogen species of this genus are described and discussed. The former, are mainly based in PCR amplification of a concrete DNA fragment and posterior confirmation with Southern-blot; RAPD techniques and less frequently microsatellite markers analysis and DNA-DNA hybridization to a moderately repeated, inactive, retrotransposon-like DNA Afut1 are used in typing clinical isolates.     

古代DNA研究实验技术

现代分子生物技术的发展,使从古代样品中获取微量DNA成为现实。在过去的十多年里,古DNA研究取得了重大进展,但实验方案还需要加以改进,其结果的分析与推论也需要多方面的验证。本综述着重介绍了古DNA研究的实验技术及可靠性分析。 Experimental Techniques for Ancient DNA Research YANG Shu-juan1,LAI Xu-long1,2,TANG Xian-hua1,SHENG Gui-lian1,2 1.Faculty of Earth Sciences,China University of Geosciences,Wuhan,430074,China; 2.Institute of Life Sciences,China University of Geosciences,Wuhan,430074,China Abstract:The development of modern molecular biological techniques makes it possible to study minimum DNA from ancient materials.During past decade,a lot of significant achievements on ancient DNA research have been made in many fields especially in molecular evolutionary biology.The nature of degradation and contamination of ancient DNA from ancient biological materials pose a dominating problem in ancient DNA research.Therefore,the experiments should be modified based on the modern molecular techniques and more factors should be considered when the results are analyzed.In this paper,authors review the general experimental protocols on sampling,extraction and amplification as well as authenticity of ancient DNA. Key words：ancient DNA;authenticity;ancient DNA techniques     

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.     

A technique for the rapid extraction of microalgal DNA from single live and preserved cells

Molecular methods are increasingly being used in the study of harmful microalgae; however, DNA extraction techniques have imposed limitations on the species and questions studied, with research primarily restricted to cultured specimens. Here we describe a simple method that merges two existing techniques for DNA extraction from live and preserved single dinoflagellate cells. DNA was successfully isolated from live single cells of Gambierdiscus toxicus Adachi et Fukuyo, 1979 and cells preserved using formalin/methanol fixation. This method supplements existing techniques and expands the scope of genetics studies conducted on dinoflagellates to include routine molecular analysis of single cells isolated from field samples.     

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.     

几种主要分子生物学技术在真菌生态学研究中的应用

真菌在自然界的物质循环与降解等生态过程中发挥着重要的作用,是生态系统的重要组成部分。然而约90%的真菌种类仍然未知,且大部分难于分离和培养。因此核酸杂交;核酸序列分析;DNA指纹分析等分子生物学技术被用于真菌分类、鉴定、种群结构、群落多样性研究。本文综述了这几种主要分子生物学技术的基本原理及其在真菌生态学研究中的应用现状。     

See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of DNA molecules have been improving constantly. The combination of both techniques has appealed to researchers ever since single-molecule measurements have become possible and indeed first implementations of such combined approaches have proven useful in the study of several DNA-binding proteins in real time. Here, we describe the technical state-of-the-art of various integrated manipulation-and-visualization methods. We first discuss methods that allow only little control over the DNA conformation, such as DNA combing. We then describe DNA flow-stretching approaches that allow more control, and end with the full control on position and extension obtained by manipulating DNA with optical tweezers. The advantages and limitations of the various techniques are discussed, as well as several examples of applications to biophysical or biochemical questions. We conclude with an outlook describing potential future technical developments in combining fluorescence microscopy with DNA micromanipulation technology.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

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.     

IHF and HU: flexible architects of bent DNA

The energetic cost of bending short segments of DNA is very high. This bending is critical for the packaging of DNA and is exploited to regulate many cellular processes. In prokaryotes, IHF and HU are key architectural proteins present at high concentrations. New protein-DNA co-crystal structures, and the adaptation of advanced biophysical and biochemical techniques have led to an improved understanding of how these proteins interact with DNA. These techniques include time-resolved synchrotron X-ray footprinting, differential scanning calorimetry, isothermal titration calorimetry and single-molecule experiments.     

Ploidy levels in nonneoplastic and neoplastic thyroid cells

DNA levels in nonneoplastic and neoplastic human thyroid cells were studied by slide-cytophotometric and flow-cytophotometric techniques. Normal epithelial thyroid cells and cells from nonneoplastic thyroid disorders, i.e., toxic goiter, colloid goiter and chronic lymphocytic thyroiditis, had euploid (diploid, polyploid) DNA levels. The same was the case for benign tumors. Malignant tumors had either euploid DNA levels, indistinguishable from those in nonneoplastic and benign neoplastic cell populations, or aneuploid DNA levels. Taking into account the histopathologic diagnosis, clinical course and DNA level, the results indicate that DNA measurements in thyroid gland lesions are of limited diagnostic help but may contribute valuable prognostic information. To obtain representative and accurate DNA measurements, combined slide-cytophotometric and flow-cytophotometric techniques should be used.