小鼠细胞因子相关基因表达检测寡核苷酸芯片的制备及分析

生物芯片技术用于基因表达谱研究是近年来发展起来的一项新技术 ,该方法本质上是基于对一玻璃片或膜表面上固定的ｃＤＮＡ或寡核苷酸的分子杂交 ,这一新技术可同时测定成千上万个基因的作用方式 ,几周获得的信息用其它方法可能要几年才能得到 ,是以定量方式同时监测大量基因相对表达的强有力的新方法[1 ,2 ] 。国内外目前主要采用ｃＤＮＡ芯片进行基因表达的检测 ,芯片制备所用的ＤＮＡ探针一般为已知基因ｃＤＮＡ克隆的ＰＣＲ扩增产物或ＥＳＴ的扩增产物[3～ 8] 。对基因的表达检测来说 ,ｃＤＮＡ芯片技术是一条非常适用的检测方法 ,但在有…     

抗体和寡核苷酸双标记纳米金生物探针的制备及性能分析

基于纳米金粒子与抗体静电吸附作用,与硫醇修饰的寡核苷酸共价结合,建立一种新的双标记纳米金生物探针的制备方法.通过透射电镜（TEM）、紫外光谱、斑点免疫金渗滤法、免疫金银染色光镜观察法、荧光标记法等检测探针表征,及表面抗体活性情况和寡核苷酸的覆盖率,同时采用变性聚丙烯酰胺凝胶电泳（PAGE）检测寡核苷酸的存在.结果表明,纳米金粒子同时连接抗体和寡核苷酸后生物性能良好,且每个纳米金粒子（10±3）nm表面可覆盖寡核苷酸(92±20)条,双标记纳米金生物探针的制备具有简捷、稳定的特点.可作为一种新型探针应用于超微量蛋白质检测.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

Detection of rare mRNA species in a complex RNA population by blot hybridization techniques: a comparative survey

The detection of very rare mRNA species in a complex RNA preparation by current RNA blotting techniques is not straightforward. To be able to determine the size of mRNA molecules representing 10(-6) to 10(-7) of the total mass of an RNA preparation, a quantitative comparison of the level of detection of denatured mRNA species electrophoretically separated on agarose gels, followed by transfer to either nitrocellulose or diazobenzyloxymethyl (DBM) paper and hybridization to specific cDNA probes was carried out. Different transfer procedures were analyzed. Optimal conditions have been found which allowed the detection of RNA bands containing as little as 5 pg of a specific sequence within a few days of autoradiography following hybridization with highly labeled [32P]cDNA probes. Using this procedure it was shown that the low amounts of alpha-fetoprotein (AFP) mRNA sequences present in adult rat liver are mature AFP mRNA molecules.     

邻位连接技术：一种新的高灵敏度蛋白质检测技术

蛋白质组学(proteomics)诞生以来,高效准确的蛋白质检测技术受到越来越多的关注.最近 ,一种高灵敏度的蛋白质检测技术,邻位连接技术(proximity ligation assay, PLA)被建 立.该技术采用核酸适体(aptamer)或单/多克隆抗体 核酸复合物作为邻位连接探针(proximity probes).当一对邻位探针同时识别同一个目标蛋白分子时,它们将在空间位置上相互临近,通过连接反应形成一段可扩增的DNA标签序列,该标签序列能够反映待测蛋白的种类及浓度.该技术将对蛋白质的检测转变为对DNA核酸序列的检测,实现了特殊蛋白质的检测,定量及定位.文章从该方法的产生背景,发展过程,原理以及探针制备等方面对该方法进行了系统的介绍,列举了该方法的几种重要应用,并对该方法在蛋白质组学研究领域的应用前景进行了展望.     

Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5 h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.     

特异性的蛋白小分子荧光探针及其标记技术

活体蛋白荧光标记技术已经被广泛应用于蛋白质功能的可视化研究中。荧光蛋白常被用来研究蛋白质在生物体内的表达和定位,但由于它本身体积比较大,往往会影响目标蛋白的生物活性。特异性的小分子荧光探针以其体积小、膜透性好、背景噪音低以及制备方便的优点成为蛋白质研究的一个有力工具。本文将简要介绍近几年来各类特异性小分子蛋白荧光探针的研究进展。     

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival, as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multiclone and multicolor mapping experiments do not generate additional information. Our pooling protocol, described here with examples from thyroid cancer research and PGD, accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as 3 to 4 days. (J Histochem Cytochem 57:587–597, 2009)     

植物木质部压力探针测定技术与方法

木质部压力探针技术是目前直接测定植物木质部导管负压的唯一手段。在结构上,木质部压力探针测定系统由精密操作装置、压力探针系统和信号采集—传输一显示系统三大部分组成。其测定原理是将毛细管探针刺入木质部导管,通过传导介质将木质部导管负压传至压力传感器,压力传感器感应压力并将压力信号输出。本文从玻璃毛细管探针的制作、去气泡水的制备以及压力探针的校准、安装、测定等方面详细介绍了木质部压力探针的使用方法和注意事项。     

Comparison and Evaluation of Silver Probe Preparation Techniques for Tip-Enhanced Raman Spectroscopy

In this work, the different procedures for the fabrication of Ag probes for tip-enhanced Raman spectroscopy (TERS) in a top illumination/detection setup are proposed and tested. We focus on technologically simple methods allowing Si tips coated with plasmonic silver nanostructures and bulk metal Ag tips with good shape reproducibility to be produced for atomic force microscopy (AFM) feedback setup. The preparation of Ag TERS probes was based on chemical deposition and vacuum sputtering of Ag on the tips of commercially available Si cantilevers. A straightforward technique for the fabrication of bulk metal Ag probes by the electrochemical etching of Ag microwires was also proposed. Chemically coated, sputtered, and electrochemically etched TERS tips were characterized by scanning electron microscopy (SEM). The produced tips were tested for TERS measurements using graphene oxide (GO) as the target analyte in a top illumination setup. A comparative analysis of enhancement factors (EF) for the different types of tips (probes) is presented in this work.     

Identification and characterization of proteins binding A + U-rich elements

A + U-Rich elements (AREs) have been extensively investigated as cis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to the study of any protein displaying sequence-specific RNA binding activity. The techniques described here include the purification of native AUF1 from cultured cells as well as the preparation of recombinant AUF1 proteins using a bacterial expression system. Analyses of RNA-protein interactions are also described, including the use of gel mobility shift assays with synthetic RNA probes to monitor specific RNA binding activity in cell extracts or with recombinant proteins. Variations of this technique are also described to evaluate the RNA binding affinity of recombinant proteins and the use of specific RNA competitors to assess RNA determinants of protein binding specificity. Other techniques presented include the identification of specific proteins in RNA:protein complexes using antibody supershifts and the estimation of molecular weights of RNA-binding proteins by UV crosslinking. Results of individual experiments are presented as examples of some techniques. Throughout the article, suggestions are included to avoid commonly encountered problems and to assist in the optimization of these techniques for the study of other RNA-binding proteins.     

Oligo- and polynucleotide probes. A molecular hybridization method

In this review the main principles and possibilities of a hybridization analyses as a highly specific methods for diagnostics of infections and genetic diseases are discussed. Special attention is paid to the chemical approaches to the preparation of non-radioactive probes based on synthetic oligonucleotides and polynucleotides containing biotin, Eu3+ and enzyme labels. Some versions of the hybridization analysis technique are discussed.     

基因芯片技术和蛋白质组技术在神经科学中的应用及其研究进展

基因芯片技术和蛋白质组技术是最近发展起来的高通量技术,二者的出现使同时分析神经系统的大量基因的表达和基因产物蛋白质及其相互作用网络成为可能。它们在神经科学中的应用为了解脑功能提供了前所未有的机会。一个典型的基因芯片实验包括：芯片的准备或购买、靶DNA和探针的准备或标记、标记探针与靶DNA的杂交、芯片扫描和影象信息的数据分析。蛋白质组技术较为复杂,包括蛋白质分离、鉴定和信息分析三方面的内容。其中,分离技术多种多样。若分离技术以二维电泳为基础,则该实验通常由以下步骤组成：蛋白质样品的准备、电泳分离、染胶、分离蛋白点的切除、蛋白质的酶解(常用胰蛋白酶)、质谱分析(鉴定)和数据的信息处理。本文综述这两项技术的内容和实验步骤,然后着重叙述它们在神经科学中的应用,讨论其优缺点和面临的挑战,展望其发展前景。     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

A cosmid vector for systematic chromosome walking

We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.