电镜技术在核酸分子杂交研究中的应用

电子显微镜是目前唯一能直接看到DNA或RNA分子的工具,而电泳、同位素标记等技术只能间接地测定这些分子,电镜就是以它这种得天独厚的优点在核酸分子研究中崭露头角。基因的准确定位,核酸复制模型的完善,真核基因DNA倒转重复序列“十字架”结构的观 察及内含子的二发现等等,近四十年来,核酸分子杂交技术取得如此可喜的成绩,电镜所作的贡献是不能低估的。     

核酸电镜术简介

当前在核酸分子的生物化学方面已有比较深入的研究,而用电子显微镜来观察核酸分子及其变化的工作正在不断发展,这方面的研究以及技术上的改革层出不穷。限于篇幅,这里只能结合自己的工作将核酸电镜的一些主要方法和用途介绍一下。Ferguson和Davis(1978)     

引致河蟹颤抖病的病毒的核酸定性

对河蟹(中华绒螯蟹)"颤抖病"病毒及其核酸进行了电镜观察.纯化病毒经负染后由电镜观察显示,病毒粒子呈球状,有囊膜和纤突,直径约为150nm.纯化病毒经核酸酶降解杂核酸后,加入8mol/L尿素释放病毒核酸,经水相法展开后,用覆有碳膜的铜网取样,再经染色和真空镀膜,于电镜下观察.病毒核酸呈单股线状,底片经光学精确放大后,测定了核酸分子的长度,根据经验公式计算出病毒核酸分子量为5.05×106.抽提的病毒核酸在0.8%琼脂糖凝胶电泳中呈单一条带,大小为15～16kb,经核酸酶酶切显示,核酸对DNase I不敏感,对RNase、Mung Bean Nuclease敏感,根据以上特性判断,该核酸为ssRNA.     

引致河蟹颤抖病的病毒的核酸定性

对河蟹(中华绒螯蟹)“颤抖病”病毒及其核酸进行了电镜观察。纯化病毒经负染后由电镜观察显示,病毒粒子呈球状,有囊膜和纤突,直径约为150nm。纯化病毒经核酸酶降解杂核酸后,加入8mol/L尿素释放病毒核酸,经水相法展开后,用覆有碳膜的铜网取样,再经染色和真空镀膜,于电镜下观察。病毒核酸呈单股线状,底片经光学精确放大后,测定了核酸分子的长度,根据经验公式计算出病毒核酸分子量为5.05×106。抽提的病毒核酸在0.8%琼脂糖凝胶电泳中呈单一条带,大小为15~16kb,经核酸酶酶切显示,核酸对DNaseI不敏感,对RNase、MungBeanNuclease敏感,根据以上特性判断,该核酸为ssRNA。     

核酸的电镜细胞化学实用方法

核酸是生命活动中最重要的基本成份之一。鉴定或定位超微结构上的核酸,在生物学研究中具有重要意义。本文介绍了应用电镜技术在生物超微结构上鉴定或定位核酸的若干研究方法。根据所确定成份的不同,这些方法可分为三类。第一类是对总核酸的鉴定,第二类是对DNA特异地染色鉴定;第三类是对RNA选择性染色鉴定。     

环境水体中肠道病毒核酸检测方法的应用

水中病毒的检测常采用细胞培养，酶免疫及免疫电镜等技术。随着肠道病毒分子生物学研究的进展，敏感，特异和快速的病毒核酸检测方法得到广泛应用，扩展了水中病毒检测的手段。     

南瓜和丝瓜韧皮部伤流液中核酸的存在和鉴定

利用化学分析结合电镜技术,对南瓜和丝瓜韧皮部伤流液中的DNA和RNA,进行了定性和定量的研究。核酸样品经日立双波长和双光束分光光度计测定,在258 nm左右有一吸收高峰,低峰在230 nm附近,呈现出核酸的紫外吸收光谱曲线。南瓜伤流液中的DNA含量为60～238μg/ml,RNA为30～80μg/ml,而丝瓜则分别为30～120μg/ml和20～40μg/ml.DNA水解物经高压液相色谱(HPLC)测定、可将T.G.C.A四个碱基明显分开。DNA电镜观察,得到清晰的图象。电镜细胞化学鉴定,也表明伤流液中有核酸存在。上述结果证实瓜类韧皮部伤流液中含有DNA和RNA,它们可能参与了韧皮部的运输,并可随伤流液的流动而迁移。     

家蚕核型多角体病毒突变株的研究——Ⅱ.空斑法克隆及限制酶酶解分析

本研究工作用空斑技术纯化了遗传上均一的家蚕核型多角体病毒大方块包涵体株—NPVmt,而且用这种方法可以精确的进行病毒定量。通过病理学、血清学、电镜、核酸呈色反应以及NPVmt-DNA的核酸内切酶EcoRI、XbaI、BamHI的分析,在同条件下与Bm SNPV作了比较,表明NPVmt不仅包涵体突变成大方块形,而且在基因组的结构水平上与Bm SNPV有显著差异。     

植物超微结构中核酸的细胞化学方法比较研究

随着电子显微镜技术的广泛应用,植物超微结构的研究日益与生理功能密切地结合起来。为了在超微结构水平上对某种特殊的化学成分(如核酸、蛋白质、酶或多糖等)鉴定或定位,发展了诸如酶的细胞化学、核酸的细胞化学等各种细胞化学方法。电镜的细胞化学尚处于初期阶段,对植物组织尤其如此,已用于动物组织方面的许多化学反应不能用于植物材料。核酸的细胞化学方法已有许多报导,但都是以动物组织为材料发     

小鹅瘟病毒核酸链型与结构蛋白分析

采用简易方法从鹅胚中分离纯化的小鹅瘟病毒(GPV)样品相当纯净,以致在电镜下呈晶格排列。用负染色技术观察病毒的形态结构,病毒直径为18～20nm,无囊膜。病毒颗粒含有4种结构蛋白,其分子量分别为88、78、66和51kd。根据病毒的核酸链型分析,小鹅瘟病毒含有单链核酸。此结果支持了小鹅瘟病毒属于细小病毒属的结论。     

Synthesis of aminoglycoside-DNA conjugates

Development of aminoglycoside-nucleic acid conjugates is presented. Synthesis of a DNA dimer covalently linked to kanamycin and neomycin isothiocyanates has been carried out. The development of such conjugates will help couple the sequence specificity of nucleic acids to the electrostatic/shape complementarity of aminoglycoside antibiotics in binding nucleic acid targets.     

Modeling nucleic acids

Nucleic acids are an important class of biological macromolecules that carry out a variety of cellular roles. For many functions, naturally occurring DNA and RNA molecules need to fold into precise three-dimensional structures. Due to their self-assembling characteristics, nucleic acids have also been widely studied in the field of nanotechnology, and a diverse range of intricate three-dimensional nanostructures have been designed and synthesized. Different physical terms such as base-pairing and stacking interactions, tertiary contacts, electrostatic interactions and entropy all affect nucleic acid folding and structure. Here we review general computational approaches developed to model nucleic acid systems. We focus on four key areas of nucleic acid modeling: molecular representation, potential energy function, degrees of freedom and sampling algorithm. Appropriate choices in each of these key areas in nucleic acid modeling can effectively combine to aid interpretation of experimental data and facilitate prediction of nucleic acid structure.     

中温淡盐法制备低核酸酵母抽提物的工艺研究

通过建立一种新的酵母核酸中温淡盐去除法,成功研制出品质优良的低核酸酵母抽提物。工艺简单,处理时间短,温度不超过70℃,可避免高温对酵母营养物质的破坏;该法氯化钠用量少,无需脱盐处理,易产业化及应用推广;研究制备的酵母抽提物,核酸去除量达70%以上,色浅、肉香味鲜,适合食品加工。同时,附加得到高提取率、高纯度的核酸。中温淡盐法去除酵母核酸的技术,扩展了酵母制品的应用领域,更为尿酸偏高及痛风人群食用安全营养的酵母食品提供了解决方案。     

Predicting nucleic acid binding interfaces from structural models of proteins

The function of DNA‐ and RNA‐binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure‐based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high‐resolution three‐dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I‐TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high‐resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I‐TASSER produces high‐quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low‐resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

重组酶聚合酶扩增技术：一种新的核酸扩增策略

重组酶聚合酶扩增 (recombinase polymerase amplification, RPA)是近年来兴起的一种等温核酸扩增技术,它比聚合酶链式反应(polymerase chain reaction, PCR)及其它等温扩增技术更快速、便捷、高效。本文将详细介绍RPA这项新颖的技术,并对其在医疗诊断、农业、食品、生物安全等方面的研究及应用进展进行综述。期望这项技术得到更多的关注,使其发展更加完善,将来在更多的领域充分发挥作用,甚至书写核酸检测历史新篇章。     

Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. Accepted: 9 June 1999     

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic beta-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin-biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.     

A sense of closeness: protein detection by proximity ligation

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.     

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.