An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes

A label-free electrochemical method for the detection of DNA-PNA hybridization using a water-soluble, ferrocene-functionalized polythiophene transducer and single-stranded PNA probes on the nanogold modified electrode is investigated. Nanogold modified electrodes can largely increase the immobilization amount of ss-PNA capture probe and lead to an increase of the electrical signal. The ferrocene-containing cationic polythiophene do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after DNA-PNA hybridization, cationic polythiophene is adsorbed on the DNA backbone, giving a clear hybridization detection signal in differential pulse voltammetry (DPV). Very good discrimination against non-complementary DNA and four-base mismatch DNA is observed. These studies show that the proposed method can provide an alternative for expanding the range of detection methods available for DNA hybridization.     

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.     

Immobilization of homooligonucleotide probe layers onto Si/SiO(2) substrates: characterization by electrochemical impedance measurements and radiolabelling

Radiolabelling and electrochemical impedance measurements were used to characterize the immobilization of single stranded homooligonucleotides onto silica surfaces and their subsequent hybridization with complementary strands. The immobilization procedure consists of grafting an epoxysilane onto microelectronic grade Si/SiO(2) substrates, and coupling oligonucleotides bearing a hexylamine linker onto the epoxy moiety. Radiolabelling was used as a reference method to quantify the amount of immobilized and hybridized oligonucleotides. These results show that the Si/SiO(2) substrates modified with an epoxysilane yield a surface concentration of approximately 10(11) strands/cm(2) for the immobilized oligonucleotides, after vigorous washings, and that approximately 36% of these undergo hybridization with complementary strands. The impedance measurements, which provide a direct means of detecting variations in electrical charge accumulation across the semiconductor/oxide/electrolyte structure when the oxide surface is chemically modified, show that the semiconductor's flat band potential undergoes reproducible shifts of -150 and -100 mV following the immobilization and the hybridization step, respectively. These results demonstrate that electrochemical impedance measurements using chemically modified semiconductor/oxide/electrolyte structures of this type offer a viable alternative for the direct detection of complementary DNA strands upon hybridization.     

Synthesis and hybridization properties of oligonucleotides containing 6-membered azasugar nucleotides

A modified nucleoside was synthesized with adenine and a 6-membered azasugar, and it was converted to the phosphoramidite which was used for the incorporation into oligonucleotides. The hybridization properties of the modified oligonucleotides with DNA and RNA were studied.     

Nonradioactive labeling with chemically modified cytosine tails by the polymerase chain reaction.

We developed a modified nonradioactive method for the detection of DNA. This method makes use of the polymerase chain reaction for preparation of probes; that is, a DNA fragment inserted in the polylinker region of an M13 or pUC vector is amplified with primers that have a modified cytosine tail at the 5' terminus (C-tailed primers). By this method, large amounts of labeled probes can be obtained easily. After hybridization, modified cytosine tails can be detected immunologically. DNA labeled by this method could be used in plaque hybridization. We could detect 0.05 pg of dot-blotted labeled DNA in 30 min with an enzyme-catalyzed chemiluminescence reaction.     

Perylene-Labeled Oligonucleotide as a Probe in Homogeneous Hybridization Assay

Abstract

An oligodeoxynucleotide bearing a 3′-terminal perylene-containing pseudonucleoside unit was synthesized and used as a probe in homogeneous hybridization. The fluorescence anisotropy of the perylene dye rose upon hybridization of the modified conjugate with the complementary nucleotide sequence. These results provide for designing efficacious hybridization probes.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Development of an electrochemical DNA biosensor with a high sensitivity of fM by dendritic gold nanostructure modified electrode

In this paper, dendritic gold nanostructure (DenAu) modified electrode was obtained by direct electrodeposition of planar electrode into 2.8 mM HAuCl(4) and 0.1 M H(2)SO(4) solution under a very negative potential of -1.5 V. Scanning electron microscopy was used to characterize the growth evolution of DenAu with time. The whole DNA biosensor fabrication process based on the DenAu modified electrode was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. The probe DNA immobilization and hybridization with target DNA on the modified electrode could be well distinguished by using methylene blue as an electrochemical hybridization indicator. The DenAu modified electrode could realize an ultra sensitivity of 1 fM toward complementary target DNA and a very wide dynamic detection range (from 1 fM to 1 nM).     

Nonisotopic in situ hybridization as a method for nondisjunction studies in human spermatozoa

Human spermatozoa were studied with a nonradioactive in situ hybridization method. Using a chemically modified DNA probe and immunocytochemical reactions for visualization, it was possible to obtain hybridization signals in 31 of 32 semen samples. Positive hybridization reactions, depending on cell accessibility, varied from 40% to over 90% for the different samples. Using a chromosome 1-specific DNA probe, disomy for this chromosome was found in 0.67% of all accessible sperm cells.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogues Containing Novel Phosphotriester Internucleotide Linkage

Abstract

Enhanced cellular uptake, stable and discriminating hybridization and increased stability in biological media are of particular interest for oligonucleotides of potential therapeutic application. Additionally, toxicity or immunogenicity of the oligonucleotide analogues and their biodegradation products should be minimized by minimal alteration of the biological structure and effort and cost of bulk production should be as low as possible by using a standard automated synthesis protocol. Oligonucleotide phosphotriesters with oligoethyleneglycol substituents show promise to ideally combine all these advantages. Here we describe the hybridization properties and the stability of modified oligonucleotides containing triester internucleotide linkages substituted with α,ω-dihydroxy-(3,6-dioxa)-octan-1-yl group (“triethyleneglycol triester linkages”) towards enzymatic degradation. The triester linkages are stable towards exo- and endonucleases. Regardless of number and position of triester linkages, the modified oligonucleotides showed practically no decrease of T_m in hybridization studies with complementary biological oligonucleotides. In further enzymatic studies the modified oligonucleotides were highly stable towards nucleases in human blood serum.     

Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage

A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 ″ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

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.     

A survey of biochemical variants found in groundfish stocks from the North Pacific and Bering Sea

This report provides information on biochemical variants found in 17 species. These variants may be useful in answering some of the questions facing fisheries agencies concerning stock separation, hybridization, and life histories. Further studies should provide useful information in the areas of population biology of fishes in Alaskan waters.     

A modified hybridization protocol for low-density diagnostic DNA microarrays

To promote the application of DNa microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42°C to 52°C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.