Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.     

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.     

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Filter replicas and permanent collections of recombinant DNA plasmids.

A permanent, ordered collection of 23,000 recombinant DNA plasmids containing Drosophila melanogaster DNA has been established. Simple and practical methods for storing and manipulating this collection were developed. In addition, an improved, simple and inexpensive method for making paper filter replicas of such an ordered collection and of a high density (10,000 colonies/petri dish) unordered collection was developed. These filter replicas are suitable for nucleic acid hybridization screens of recombinant DNA colinies and each filter replica can be used for many (greater than 5) successive screens. The kinetics of this hybridization reaction were examined and allow design of experiments that detect colony complementarity to a nucleic acid that is 0.5% of the hybridization probe.     

Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.     

Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models

The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.     

Iridescent virus replication: patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6

The patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6 has been examined using nucleic acid hybridization techniques. Virus-specific RNA synthesis was detected 24 hr after infection. Virus-specific DNA synthesis was detected 96 hr after infection. Host-specific nucleic acid synthesis declined throughout infection, and host-specific nucleic acid synthesis was detected only in the first 48 hr of infection. The synthesis of iridescent virus progeny DNA molecules precedes the appearance of mature iridescent virus particles.     

Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.     

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.     

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.     

Specific Neisseria gonorrhoeae DNA-probes derived from ribosomal RNA

Eighteen sequences complementary to less-conserved regions within the 16S and 23S ribosomal ribonucleic acid (rRNA) of Neisseria gonorrhoeae were subcloned or chemically synthesized and used as probes in a dot-spot deoxyribonucleic acid (DNA): DNA hybridization format. Some of these probes exclusively detected Neisseria gonorrhoeae nucleic acid, whereas others also showed hybridization signals with nucleic acid from other bacterial species. Our results indicate that rRNA-derived DNA-probes can be used to differentiate between very closely related taxa without the use of Southern-blot analysis.     

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae)

Summary This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.     

Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences

Formamide is the preferred solvent to lower the melting point and annealing temperature of nucleic acid strands in in situ hybridization (ISH). A key benefit of formamide is better preservation of morphology due to a lower incubation temperature. However, in fluorescence in situ hybridization (FISH), against unique DNA targets in tissue sections, an overnight hybridization is required to obtain sufficient signal intensity. Here, we identified alternative solvents and developed a new hybridization buffer that reduces the required hybridization time to one hour (IQFISH method). Remarkably, denaturation and blocking against repetitive DNA sequences to prevent non-specific binding is not required. Furthermore, the new hybridization buffer is less hazardous than formamide containing buffers. The results demonstrate a significant increased hybridization rate at a lowered denaturation and hybridization temperature for both DNA and PNA (peptide nucleic acid) probes. We anticipate that these formamide substituting solvents will become the foundation for changes in the understanding and performance of denaturation and hybridization of nucleic acids. For example, the process time for tissue-based ISH for gene aberration tests in cancer diagnostics can be reduced from days to a few hours. Furthermore, the understanding of the interactions and duplex formation of nucleic acid strands may benefit from the properties of these solvents.     

DNA生物传感器在环境污染监测中的应用

基于生物催化和免疫原理的生物传感器在环境领域中获得了广泛应用.近年来,随着分子生物学和生物技术的发展,人们开发了以核酸探针为识别元件,基于核酸相互作用原理的DNA生物传感器.该传感器可用于受感染微生物的核酸序列分析、优先控制污染物的检测以及污染物与DNA之间相互作用的研究,在环境污染监测中具有潜在的巨大应用前景.简要介绍了核酸杂交生物传感器的基本原理及其在环境微生物和优先控制污染物（priority pollutant）检测中的应用研究进展.     

Thermodynamics of DNA-RNA heteroduplex formation: effects of locked nucleic acid nucleotides incorporated into the DNA strand

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue exhibiting enhanced hybridization efficiency toward complementary strand. The potential of LNA-based oligonucleotides has been sought to improve the selectivity and specificity of probe sets employed in detection and specific targeting of nucleic acids. We have evaluated the influence of "locked nucleic acid" residues on hybridization thermodynamics, counterions and hydration of DNA.RNA heteroduplex using spectroscopic and calorimetric techniques. One to three LNA substitutions have been introduced either at the adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) residues of the DNA strand. A complete thermodynamic profile for heteroduplex formation suggested that LNA-induced stabilization results from a favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, DeltaCp, accompanying the heteroduplex formation. Isothermal titration calorimetry measurements indicated an increase in binding affinity of the two strands as the LNA content of the heteroduplex is increased. Overall our result demonstrated that the effect of LNA-substitution at the thymine residue is more pronounced compared to the adenine residue. Furthermore, optical melting studies showed that, compared to an unmodified duplex, the formation of LNA-modified duplex is accompanied by a higher uptake of counterions and a lower uptake of water molecules. Our result, thus, presents a preliminary attempt toward the characterization of hybridization thermodynamics of the LNA-based probe-target sets, which will in turn aid in the selection of optimal conditions for hybridization experiments, and evaluation of the minimum probe-length required for hybridization and cloning experiments.     

Ultrasensitive electrical detection of nucleic acids by hematin catalysed silver nanoparticle formation in sub-microgapped biosensors

An ultrasensitive electrical detection method of nucleic acids has been demonstrated on sub-microgapped biosensor. In this method, peptide nucleic acid (PNA) probes were firstly immobilized in the gap areas of a pair of interdigited microelectrodes and then were hybridized with their complementary target DNA. After hybridization, hematin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hematin molecules act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Nearly 1fM sensitivity was achieved under optimal conditions. This approach is also applicable to the detection of RNA.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.