Tire footprint characteristics as a function of soil properties and tire operations

The main objective of the following presentation is to examine the possibility of predicting agricultural tire footprint parameters under different operational conditions. The experimental part of the research involved the operation of two agricultural transport tires on two soils, under variations of tire load, inflation pressures and soil moisture contents. Results obtained show that tire footprint parameters, such as contact area, length, width and sinkage, can be reliably predicted using multifactorial linear and total regressions, within the range of recommended tire loads, inflation pressures and soil moisture contents around the plastic limit.     

A new field single wheel tester

A new field single wheel testing device, as part of a field testing unit, was developed to perform traction tests on agricultural or cross country tires in the field. The tire testing device is mounted at the rear of a heavy wheeled tractor that also carries a unique soil property testing device at the front. The vertical, horizontal and side forces are measured inside a frame that holds the test wheel, while the torque is measured by a separate linkage system. The tire testing device is capable of testing tires up to 2 m in diameter; it can apply vertical force up to 50 kN, and torque up to 31 kNm.     

DNA analysis on electrophoretic microchips: effect of operational variables

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.     

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.     

GENERAL PURPOSE LAMPS INDUCE POLYOMA DNA REPLICATION IN H3 CELLS

Abstract— We have previously demonstrated the ability of UVC (254 nm) radiation to induce asynchronous polyoma replication in rat fibroblast cells (H3 line) that contain an integrated copy of polyoma virus. In the present study we show that general purpose lamps can induce polyoma replication in these cells as well. The amount of UV radiation emitted by three different light sources was determined and the effects of each source on the replication of polyoma DNA was assessed. Our findings indicate that a 100 W incandescent lamp had a minimal effect on replication, whereas a 90 s exposure to a halogen lamp or a 160 W mercury vapor lamp induced replication 1.5-fold and 2-fold, respectively, in comparison with nontreated controls. We have previously shown that asynchronous polyoma replication in H3 cells involves UV-inducible cellular protein factors. Our present results indicate that these factors are also activated by exposure to commonly used lamps that emit comparable doses of UV radiation.     

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.     

Direct haplotype detection of adjacent polymorphic sites in the regulatory region of the dopamine D4 receptor (DRD4) gene

A novel method of haplotype identification is discussed allowing simultaneous detection of two adjacent polymorphic sites, a single nucleotide polymorphism (SNP) and a length polymorphism within 1-2 kilobase distance. The method combines allele specific-amplification with high-throughput, automated ultrathin-layer gel electrophoresis analysis of fragment size polymorphism. A typical application is shown for genotyping the -521 C/T single nucleotide polymorphism and the 120 bp duplication in the 5"-upstream region of the dopamine D4 receptor (DRD4) gene. We have also demonstrated that the haplotypes of double heterozygotes for -521 C/T and for the 120 bp duplication can be clearly distinguished, that has only been possible previously by extensive pedigree analysis.     

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.     

Rapid quantification of human complement component C4A and C4B genes by capillary gel electrophoresis

Complement component 4 (C4) is an important plasma protein playing a major role in the human defense mechanism against infectious diseases and inflammatory processes. The C4A and C4B genes, encoding the two isoforms of complement 4, are located in the nuclear serine/threonine protein kinase-C4A or B gene-cytochrome 21-hydroxylase-tenascin X module (RP-C4-CYP21-TNX) and manifested by variable copy numbers among individuals between zero to six in the human diploid genome. Quantification of the C4A and C4B genes has great clinical importance since unbalanced production of C4A and C4B proteins might be associated with pathological immune processes. Albeit, high-throughput analysis methods for C4 gene dosage determination are not yet available. Here we present a novel combination of allele-specific PCR and CGE separation for rapid quantification of the C4A and C4B genes where a single-step, single-tube PCR reaction generates two allele-specific (C4A and C4B) and two control amplicons, followed by CGE analysis of the four fragments. The method presented in this paper enables automated and high-throughput gene dosage analysis of large sample cohorts.