Survival and Activity of a 3-Chlorobenzoate-Catabolic Genotype in a Natural System

A chlorobenzoate-degrading Alcaligenes strain, BR60, was introduced to flowthrough lake microcosms and exposed to 3-chlorobenzoate (3Cba) concentrations from 0 to 25 μM. A DNA probe specific for BR60 chlorobenzoate catabolic genes was used with the most probable number (MPN) technique to enumerate bacteria harboring this genetic information. This MPN-DNA hybridization method combined with [U-¹⁴C]3Cba uptake rate measurements allowed the correlation of the size and activity of a specific catabolic population in a natural mixed community for the first time. An experiment involving the release of a streptomycin-resistant strain of BR60 indicated that estimates of bacteria carrying the introduced catabolic genotype often outnumbered plate count estimates of viable BR60 by as much as 3 orders of magnitude, particularly when 3Cba inputs were high. The MPN-DNA hybridization method provided catabolic population estimates highly correlated to 3Cba exposure levels and the [U-¹⁴C]3Cba uptake rates in the microcosms. Plate counts of BR60 were poorly correlated with both 3Cba exposure levels and uptake rates. In the absence of chlorobenzoate selection, the catabolic genotype declined to very low levels by the MPN-DNA hybridization technique after 8 weeks in the microcosms.     

Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40°C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.     

Assessment of exposure to intermediate frequency electric fields and contact currents from a plasma ball

While electric fields at intermediate frequencies are not widely utilized for industrial technologies, surprisingly, certain toys emit the highest electric fields found in our living environment. These toys, plasma balls, are devices that use high voltage to create ionized light discharges. In this study, we assessed exposure to electric fields and contact/induced current from a recreational plasma ball device. The electric field strength was measured as a function of distance from the device, and the contact/induced current was measured with a current clamp in different exposure situations with point or grasping contact. The characteristic spectra of the electric field and contact current were measured, and both the multiple frequency rule and weighting of the spectra were applied according to the International Commission on Non-Ionizing Radiation Protection (ICNIRP) 1998 and 2010 guidelines. The results indicate that the recommended reference levels for the general public are exceeded at distances <1.2 m, and that the contact currents in the hand may be twice higher than recommended by the general public guidelines.     

Effects of non-uniform static magnetic fields on the rate of myosin phosphorylation

The effect on myosin phosphorylation from exposure to a magnetic field generated by an array of four permanent magnets was investigated. Two lateral positions in the non-uniform field over the array were explored, each at four vertical distances over the surface of the device. The rate of myosin phosphorylation was found to depend on the position laterally over the array as well as the distance from the device surface. The square magnet array was comprised of axially magnetized, cylindrical NdFeB permanent magnets arranged with poles of alternating polarity in a plane (MagnaBloc trade mark therapeutic device). Detailed dosimetry of the magnet array was compiled: the magnetic flux density averaged over the exposure volume spanned the range 0.7-86 mT for the eight different exposure positions. The corresponding range for the absolute field gradient was 0.4-20 T/m. Comparing the dosimetry to the experimental outcome, our results imply that magnetic field amplitude alone is not sufficient to describe the influence of the field in this preparation.     

Restriction endonuclease recognition and Southern hybridization of bromodeoxyuridine-substituted genomic DNA

Abstract. Human glioma cell lines exposed to various concentrations of bromode-oxyuridine (BrdUrd) were studied to determine the effect of BrdUrd substitution on restriction endonuclease recognition and Southern hybridization of genomic DNA. BrdUrd substitution had no effect on the recognition of restriction endonucleases. When the exposure to BrdUrd was 2 h or less and the BrdUrd substitution rate was less than 40%, there was no difference in the density of hybridized bands after Southern hybridization using human non-recombinant complementary DNA as a probe. Hybridization was suppressed significantly by exposures longer than 24 h or BrdUrd substitution rates greater than 40%. These results suggest that the BrdUrd substitution rate and the exposure time to BrdUrd influence the hybridization reaction by a DNA probe. Brief exposure (up to 2 h) to BrdUrd does not influence restriction endonuclease recognition or Southern hybridization of genomic DNA.     

Use of a radiopaque localizer grid to reduce radiation exposure

Background

Minimally invasive spine surgery requires placement of the skin incision at an ideal location in the patient's back by the surgeon. However, numerous fluoroscopic x-ray images are sometimes required to find the site of entry, thereby exposing patients and Operating Room personnel to additional radiation. To minimize this exposure, a radiopaque localizer grid was devised to increase planning efficiency and reduce radiation exposure.

Results

The radiopaque localizer grid was utilized to plan the point of entry for minimally invasive spine surgery. Use of the grid allowed the surgeon to accurately pinpoint the ideal entry point for the procedure with just one or two fluoroscopic X-ray images.

Conclusions

The reusable localizer grid is a simple and practical device that may be utilized to more efficiently plan an entry site on the skin, thus reducing radiation exposure. This device or a modified version may be utilized for any procedure involving the spine.     

Preparation of Extended Metaphase Chromosomes from Human Cultured Cells Using a Topoisomerase II Inhibitor,ICRF-193

Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.     

Preparation of extended metaphase chromosomes from human cultured cells using a topoisomerase II inhibitor, ICRF-193.

Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.     

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.     

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

A filtration, incubation and staining reactor including a new protocol for FISH

A newly developed device for performing fluorescence in situ hybridization (FISH) is described. An adapted procedure was compared with two typical FISH protocols. Tests were performed with Pseudomonas cells and the gene probe EUB338. With the novel procedure, we obtained a better recovery of cells and less variability in results.     

A miniature integrated device for automated multistep genetic assays

A highly integrated monolithic device was developed that automatically carries out a complex series of molecular processes on multiple samples. The device is capable of extracting and concentrating nucleic acids from milliliter aqueous samples and performing microliter chemical amplification, serial enzymatic reactions, metering, mixing and nucleic acid hybridization. The device, which is smaller than a credit card, can manipulate over 10 reagents in more than 60 sequential operations and was tested for the detection of mutations in a 1.6 kb region of the HIV genome from serum samples containing as few as 500 copies of the RNA. The elements in this device are readily linked into complex, flexible and highly parallel analysis networks for high throughput sample preparation or, conversely, for low cost portable DNA analysis instruments in point-of-care medical diagnostics, environmental testing and defensive biological agent detection.