Purification of nucleic acids by hybridization to affinity tagged PNA probes

The use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridization. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.     

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.     

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.     

Evaluation of single-stranded nucleic acids as carriers in the DNA-directed assembly of macromolecules

Current developments in nanosciences indicate that the self-assembly of macromolecules, such as proteins or metallic nanoclusters, can be conveniently achieved by means of nucleic acid hybridization. Within this context, we here report on the evaluation of single-stranded nucleic acids to be utilized as carrier backbones in DNA-directed self-assembly. A microplate solid-phase hybridization assay is described which allows rapid experimental determination of the hybridization efficiencies of various sequence stretches within a given nucleic acid carrier strand. As demonstrated for two DNA fragments of different sequence, the binding efficiencies of several oligonucleotides depend on the formation of specific secondary structure elements within the carrier molecule. A correlation of sequence-specific hybridization capability with modeled secondary structure is also obvious from experiments using the fluorescence gel-shift analysis. Electrophoretic studies on the employment of helper oligonucleotides in the formation of supramolecular conjugates of several oligonucleotide-tagged proteins indicate, that structural constraints can be minimized by disruption of intramolecular secondary structures of the carrier molecule. To estimate the influences of the chemical nature of the carrier, gel-shift experiments are carried out to compare a 170mer RNA molecule with its DNA analogue. Ternary aggregates, containing two protein components bound to the carrier, are formed with a greater efficiency on the DNA instead of the RNA carrier backbone.     

Nucleic acid hybridization: from research tool to routine diagnostic method

The nucleic acid hybridization reaction is extremely specific and thus a valuable tool for the identification of genes or organism of interest. The increasing use of nucleic acid hybridization in applied fields like diagnostic medicine has led to the development of more convenient hybridization assays than those originally used in basic research. In conventional nucleic acid hybridization methods immobilized nucleic acids are detected on a filter by a radiolabelled probe. Sandwich hybridization is a simple test format for the analysis of unpurified biological material, but has the disadvantage of a slow reaction rate. Solution hybridization methods are fast and easy to perform provided that a method to separate the formed hybrids from the reaction mixture is available. In non-isotopic detection the nucleic acid probe is modified with a chemical group, which is identified with a labelled detector molecule after hybridization. The low sensitivity of detection is the main problem in nucleic acid hybridization methods. Procedures to amplify the detectable signal or the amount of detectable nucleic acid sequences are potential solutions to this problem. The new hybridization methods have successfully been used for some applications, but still need to be combined into well performing tests to be applicable to any desired purpose.     

Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification

Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10,000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

Microbial DNA diagnostic technology

The principle objectives when creating a robust DNA diagnostic assay system are sensitivity, specificity and minimal read-time. To meet these ends, depending on the specifically defined test, various aspects of molecular hybridization methodology must be optimized. In particular, among other things, attention has focused on (i) formulating highly specific probes; (ii) devising sensitive nonisotopic detection systems, (iii) minimizing the extent of preparing clinical samples for assaying, (iv) amplifying the target sequence to augment sensitivity and (v) enhancing hybridization kinetics to speed up the reaction period. In this article, some recent studies that are directed to the development of nucleic acid hybridization systems for clinical diagnosis of microorganisms are considered.     

DNA-RNA hybridization.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.     

Diagnostic nucleic acid hybridizations for infectious diseases

The use of nucleic acid hybridization techniques has expanded into many areas, including studies of gene structure and function, routine diagnosis of human, animal and plant diseases, and also forensic science. In situ hybridization is one of the techniques currently available for nucleic acid hybridization and has some distinct advantages compared with standard techniques such as dot-blot, Southern or Northern hybridization, in which the histological structure is lost during extraction of nucleic acids. On the other hand, immunohistochemical staining is one branch of histochemistry that has received considerable attention in recent years as a very sensitive method for localization of specific proteins and other antigenic macromolecules within tissues and cells. This technique has also been widely used for clinical diagnosis and in various fields of research in medical science and biology. Automation of colorimetric in situ hybridization and immunohistochemistry would greatly contribute to the ease of introducing these techniques for routine pathological diagnosis and would improve the reproducibility of the assay. In this review, author will describe the development of an automated method for in situ hybridization and immunohistochemical staining using an automatic machine for both procedures.     

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     

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

阪崎肠杆菌核酸提取纯化方法分析比较

阪崎肠杆菌是一种食源性致病菌,目前越来越多的分子检测技术用于该菌的检测,以取代传统的检测技术。针对分子检测技术相应的核酸标准物质的研制势在必行。核酸的提取和纯化是核酸标准物质的研制过程中的重要环节之一,快速高效高质量,低毒低成本已成为核酸提取的重要目标。就现有方法进行分析比较,重点对常用的三种提取微生物基因组DNA的试剂盒进行了全方位比较,获得了阪崎肠杆菌较优的基因组DNA提取方法,并对提取的DNA进行PCR特异性扩增检测,获得较清晰的谱带。为阪崎肠杆菌核酸标准物质的研制奠定了基础。     

多彩色荧光原位杂交技术原理及其应用

多彩色荧光原位杂交是一门新兴的分子细胞遗传学技术,它用几种不同颜色的荧光素单独或混合标记的探针,进行原位杂交,同时检测间期细胞或中期细胞中的几个特异核酸序列,为分析癌症遗传不稳定性提供了一种简便、快速、可靠的方法,并广泛应用于物理图谱绘制、致突变研究、肿瘤病理学和产前诊断.     

Hybridization Analysis of Chesapeake Bay Virioplankton

It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the total virioplankton abundance (ca. 10⁴ PFU/ml) could be detected. Titers of either a single virus species or a group of viruses changed over time, increasing to peak abundance and then declining to low or undetectable levels, and were geographically localized in the bay. Peak signal intensities, i.e., peak abundances of virus strains, were 10-fold greater than the low background level. Furthermore, virus species were found to be restricted to a particular depth, since probes specific to viruses from bottom water did not hybridize with virus genomes from surface water at the same geographical location. Overall, changes in abundances of specific viruses within the virioplankton were episodic, supporting the hypothesis that viral infection influences, if not controls, clonal diversity within heterotrophic bacteria and phytoplankton communities.     

Imaging individual microRNAs in single mammalian cells in situ

MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.