Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA

A new technique is described for the rapid detection of specific nucleic acid sequences in unamplified DNA samples. The method consists of using two nucleic acid probes complementary to different sites on a target DNA sequence. The two probes are each labeled with different fluorescent dyes. When mixed with a sample containing the target DNA, the two probes hybridize to their respective binding sites on the same target DNA molecule. The sample is then analyzed by a laser-based ultrasensitive fluorescence system capable of detecting single fluorescent molecules at two different wavelength channels simultaneously. Since the probes are bound to the same target DNA molecule, their signals appear simultaneously. Thus, coincident detection of both dyes provides the necessary specificity to detect an unamplified, single-copy target DNA molecule in a homogeneous assay. If the target is not present, only uncorrelated events originating from free probes will be observed at either channel. Phage lambda DNA in a background of salmon genomic DNA was detected as a two-dye coincident signal at a relative concentration of one lambda molecule per salmon genome. In a control sample, cleavage of the lambda DNA between the two probe binding sites eliminated the coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applications to genetic analysis are discussed.     

Simultaneous multiple analyte detection using fluorescent peptides and capillary isoelectric focusing

Von Frey filaments used for testing mechanical thresholds are mechanically unstable and their use is difficult to standardize. We have therefore constructed a hand-held electronic pressure algometer. The pressure algometer is connected to a computerized data collection system, allowing on-line display of the applied force as well as the application rate. Data stored on the computer can be replayed and further analyzed. Using this apparatus, we have measured the pressure-induced withdrawal thresholds in rats with surgically induced neuropathy. The probe, with a circular tip of 1.0 mm diameter, was applied manually with a pressure increasing by approximately 0.05 N/s. Presurgical thresholds were normally distributed with a mean of 0.415 N, showing no significant difference between paws. During 2 weeks after surgery, the thresholds of the operated side were significantly reduced (range, 0.209-0.318 N), while the thresholds of the non-operated side remained at higher values (range, 0.432-0.491 N). Thresholds of control rats without surgery were in the 0.380-0.520 N range, with no significant difference between paws. In an additional experiment it was shown that interobserver reliability was high, both between withdrawal threshold values obtained and between rates of application used. In conclusion, the electronic algometer allows standardization of testing, detailed documentation of each experiment and provides an objective and accurate method for measuring the reactions of test animals to mechanical stimuli.     

A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences

Chronic hepatitis caused by the hepatitis C virus (HCV) is a common condition that leads to cirrhosis and hepatocellular carcinoma. Current treatment with interferon is unsatisfactory, with a low percentage of patients who respond and uncertain high-term significance; in addition, it is associated with sometimes severe side effects. The increasing sophistication of molecular biology has enabled viral characteristics such as viral load, genotypes, and quasi-species to be identified, which may help predict a patient's response to interferon treatment. We suggest that interferon therapy for hepatitis C virus should be restricted to referral centers in the context of controlled trials.     

Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5' noncoding region for detection of bovine viral diarrhea virus

Primers and probes derived from conserved sequences located in the 5' noncoding region of pestiviruses were evaluated for detection of bovine viral diarrhea virus. With these reagents, hybridization and polymerase chain reaction tests detected 62 of 90 and 90 of 90 bovine viral diarrhea virus isolates, respectively. A quick lysis method for preparing RNA for use in polymerase chain reaction amplification also was evaluated.     

Hybridization of peptide nucleic acid

The thermodynamics of hybridization and the conformations of decameric mixed purine-pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 degreesC increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (DeltaTm = -12 degreesC) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA.     

Recent development of nucleic acid drug

The dynamics of ovarian follicular development and the pattern of pituitary and ovarian hormone concentration were investigated during the luteal phase in ewes with autotransplanted ovaries. The follicles were measured by ultrasound and samples of ovarian and jugular venous blood were collected at intervals of 12 h. Blood samples were collected before and after a GnRH challenge (250 ng GnRH, i.v.) to allow the determination of basal and LH-stimulated concentration of ovarian steroids. Throughout the luteal phase, large antral follicles developed in three waves, each of which was preceded by a rise in the concentration of FSH (P < 0.05). The concentrations of oestradiol and androstenedione in the unstimulated and LH-stimulated samples were similar (P > 0.05) during the first 3 days of the luteal phase but differed thereafter, with the LH-stimulated being significantly higher than the basal concentrations (P < 0.05). In the first wave of follicular development the changes in follicular size were accompanied by an increase in the concentration of ovarian steroids and inhibin A. During the second follicular wave, although changes in follicle diameter were similar to the first wave (P > 0.05), the basal concentration of ovarian steroids and inhibin A remained unchanged throughout the period of emergence and demise of the large follicles. These results confirm that the development of large antral follicles during the luteal phase of the sheep occurs in successive waves that are associated with fluctuations in FSH secretion. However while the results strongly suggest that fluctuations in both inhibin A and oestradiol secretion control FSH during the first follicular wave, the cause of the FSH fluctuations associated with waves two and three is unclear. Final resolution of this issue may need to await the development of a specific assay for dimeric inhibin B.     

Molecular mechanics of nucleic acid sequence amplification

Protocols for in vitro amplification of nucleic acids are proliferating and there are now several methods that will contribute both to genetic research and to the diagnosis of a wide range of diseases. Here, I present the working principles of some of these molecular machines for amplifying DNA or RNA and discuss the lines along which new methods of amplifying nucleic acids may be developed.     

Antisense nucleic acid therapy of influenza virus

We have demonstrated that Antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Liposomal preparations of oligonucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier. In the observation of the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODN-PB2-as treated clone 76 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODN-PB2-as accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed on the endosomes and in the cytoplasmes of the free antisense phosphorothioate oligonucleotides treated clone 76 cells.     

Simultaneous leaching of a deep-sea manganese nodule and chalcopyrite in hydrochloric acid

Hydrochloric acid leaching of chalcopyrite and a manganese nodule, in combination, was studied using powder samples. Chalcopyrite, which does not dissolve well in HCl, was effectively leached in the presence of a manganese nodule at 3 to 4 M HCl. The rates of dissolution of metal values from the nodule were also enhanced in the presence of chalcopyrite. Dissolution was found to occur through three routes: (1) the galvanic interaction between CuFeS₂ and MnO₂, (2) the action of the Fe³⁺/Fe²⁺ redox couple, and (3) the action of Cl₂ gas generated from the MnO₂-HCl reaction on CuFeS₂. The last route appeared to make the major contribution to the dissolution. The combined leaching of a nodule and chalcopyrite appears to be promising from both technical and economic points of view.     

Hydrogen bonding of amino acid side chains to nucleic acid bases

Absorption studies of hydrogen bonding association between nucleic acid bases and amino acid side chains show that association constants can be determined from difference absorption spectra in cyclohexane and chloroform. Association constants for the binding of 9-ethyladenine, 1-cyclohexyluracil and 1-cyclohexylcytosine to side chains of serine, threonine, aspartic acid, lysine, cystéine, methionine and tyrosine are reported. Results obtained in chloroform and cyclohexane are compared.     

Novel DNA detection system of flow injection analysis (2). The distinctive properties of a novel system employing PNA (peptide nucleic acid) as a probe for specific DNA detection

In order to realize immediate detection of a double stranded DNA amplified by Polymerase Chain Reaction (PCR), we applied Peptide Nucleic Acid (PNA) to the probe of DNA detection system using Surface Plasmon Resonance (SPR). We report our success in immediate detection of PCR products solution with high sequence-specificity.     

Metabolism of caffeine to nucleic acid precursors in mammalian cells

The mission of this study was to determine whether or not arteriovenous connections, indicative of a "closed" type of circulation, existed in the human spleen. Spleens from four patients requiring therapeutic splenectomy were the basis for this report. Scanning electron microscopy of plastic corrosion casts, prepared from these four spleens, revealed direct vascular conduits between splenic pulp arteries or arterial capillaries and the venous sinuses in the red pulp. Also demonstrated were a few arteriovenous shunts between pulp arteries or arterial capillaries and pulp or trabecular veins. Inclusion of sized microspheres in low-viscosity perfusion plastic illustrated that some diameters of the connecting shunts were 7-10 mum, with other shunts even smaller. Not only do arteriovenous connections exist in human spleens, but their frequency, as revealed by methods accentuating three-dimensional aspects of the splenic microcirculation, justify future reconsiderations of the functional significance of this closed type of circulation. Examination of samples of the same intact spleens, prepared by freeze-fracture and conventional critical-point drying, also revealed an "open" type circulation structure, namely, pore-patterned sinus walls that could facilitate blood cell movement from pulp cords into venous sinuses. Scanning electron microscopy thus has provided direct evidence that human spleens have both "open" and "closed" circulatory pathways in their microvasculature.     

Molecular modelling study of the netropsin complexation with a nucleic acid triple helix

The relationship between lipid peroxidation and uptake of transferrin- free iron, Fe(II), by reticulocytes in an experimental system for studying membrane transport of Fe(II) was investigated by using free radical scavengers: BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), superoxide dismutase, alpha-tocopherol, propyl gallate and DPPD (N,N-diphenyl-1,4-phenylenediamine), and producers: t-butyl hydroperoxide, cumene hydroperoxide, H2O2 and aluminium carbonate. Measurements were made of MDA (malondialdehyde) and the rate of Fe(II) uptake from a sucrose solution buffered at pH 6.5 by Pipes. Most scavengers and producers used could increase or decrease only slightly the rate of Fe(II) uptake and some of them had no effect on Fe(II) uptake and MDA could not be detected at iron concentration of lower than 10 microM and incubation time of 20 min. At iron concentration of higher than 100 microM and incubation time of 4 h, there was the production of MDA which increased with the increment of iron concentration of incubation medium and BHT could inhibit the production of MDA. In addition, no difference was found in the rates of Fe(II) uptake in three experimental groups whose incubation medium was buffered by Pipes, Mops and Mes respectively. The results suggested that iron could induce free radical reaction under experimental conditions, especially at high concentration of iron and longer incubation time; however, at low concentration of iron (<10 microM) and the usual incubation time (20 min) free radical reaction was very slight and the extent of the reaction was not enough to damage the integrity and function of the membrane of reticulocytes, and that Fe(II) uptake by reticulocytes was not the result of free radical reaction and lipid peroxidation. It was therefore concluded that iron could not initiate its own membrane transport in rabbit reticulocytes by free radical reaction and lipid peroxidation and that the experimental system we used for studying membrane transport of Fe(II) is valid.     

Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.