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Biomedical subjects

A Guttman

Publications and source records attributed to A Guttman.

At least 19 recordsLinked to original sources
PubMed2001

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene.

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.

Alleles↗
PubMed2001

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.

DNA↗
PubMed2001

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.

Adrenal Hyperplasia, Congenital↗
PubMed2000

Rapid analysis of covalently and non-covalently fluorophore-labeled proteins using ultra-thin-layer sodium dodecylsulfate gel electrophoresis.

Gel electrophoresis is one of the most frequently used tools for the separation of complex biopolymer mixtures. In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass and protein purity. Although the method is informative, it is cumbersome, time consuming and lacks automation. In this paper we report an automated, high-performance SDS gel electrophoresis system that is based on electric-field-mediated separation of SDS-protein complexes using an ultra-thin-layer platform. The integrated fiber optic bundle-based scanning laser-induced fluorescence detection technology readily provided high sensitivity, real-time detection of the migrating solute molecules. Rapid separations of covalently and non-covalently labeled proteins were demonstrated in the molecular mass range 14,000 to 205,000 in less than 9 and 16 min, respectively. Excellent quantitation and lane-to-lane migration time reproducibility were found for all the solute components using the multilane separation platform. The limit of detection was found to be 1.5-3 ng/band for both labeling methods, with excellent linearity over a six times serial double-dilution range. Molecular mass calibration plots were compared for both covalently and non-covalently labeled proteins. A linear relationship was found between the molecular mass and electrophoretic mobility in the case of covalently labeled samples, while a non-linear relationship was revealed for the non-covalently labeled samples.

Calibration↗
PubMed2000

Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling.

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time-consuming post- or preseparation staining/labeling. By combining the multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.

Autoanalysis↗
PubMed2000

Ultra-thin-layer agarose gel electrophoresis II. Separation of DNA fragments on composite agarose-linear polymer matrices.

The effect of hydrophilic linear polymer additives (non-cross-linked polyacrylamide, hydroxyethyl cellulose and polyethylene oxide) on the migration behavior of double stranded DNA molecules, ranging from 200-1000 base pairs, were studied in ultra-thin-layer agarose gel electrophoresis. The detection sensitivity was found to be less than 0.1 ng/band using To-Pro-3 fluorophore labeling and fiber optic bundle-based scanning detection system with a 640 nm red diode laser. Among the various polymers investigated, addition of linear polyacrylamide resulted in the best separation performance (steepest Ferguson plots), while composite gels with hydroxyethylcellulose and polyethylene oxide still exhibited adequate resolving power. Using the composite matrices of 1% agarose-linear polyacrylamide (0.5-3%), 1% agarose-hydroxyethylcellulose (0.2-1%) and 1% agarose-polyethylene oxide (0.2-1%), the mechanism of the separation was found to be in the Ogston sieving regime. Activation energy curves were also plotted based on the slopes of the Arrhenius plots of the various composite matrices, and exhibited decreasing characteristics for the agarose-linear polyacrylamide composite matrix and increasing characteristics for the agarose-hydroxyethylcellulose and agarose-polyethylene oxide composite matrices.

Acrylic Resins↗
PubMed2000

Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis.

We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele-specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium-bromide-labeled fragments.

Automation↗
PubMed2000

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes.

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.

Chemical Phenomena↗
PubMed2000

Rapid and sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-layer gel electrophoresis.

Prior studies have revealed possible association between the presence of a seven repeat of the 48 bp variable number tandem repeat polymorphism of the human dopamine D4 receptor gene (DRD4) and some normal and pathological human traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorrect genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase chain reaction (PCR) products, resulting in the so-called allele dropout. In this paper we optimized the conditions for simultaneous amplification of shorter and longer amplicons of the 48 bp repeat region of the DRD4 gene in order to avoid the loss of the longer allele and consequent incorrect genotyping, using very low DNA template concentrations and partial replacement of 2'-deoxyguanosine-5'-triphosphate (dGTP) by 2'-deoxyinosine-5'-triphosphate (dITP). The optimized PCR method in combination with high throughput automated ultrathin-layer gel electrophoresis was suitable for rapid genotyping from less than a nanogram DNA using noninvasive sampling (buccal epithelial cells). All detected genotypes are presented, including such rear heterozygotes as the 2 x and 8 x 48 bp repeats in the same sample, showing the reliability of our novel detection method of longer alleles in the presence of shorter alleles.

Automation↗
PubMed2000

Ultrathin-layer gel electrophoresis of biopolymers.

Emerging need for large-scale, high-resolution analysis of biopolymers, such as DNA sequencing polymerase chain reaction, (PCR) product sizing, single nucleotide polymorphism (SNP) hunting and analysis of protein molecules necessitated the development of automated and high-throughput gel electrophoresis based methods enabling rapid, high-performance separations in a wide molecular weight range. Scaling down electric field mediated separation processes supports higher throughput due to the applicability of higher voltages, thus speeding up analysis time. Indeed, efforts in miniaturization resulted in faster, easier, less costly and more convenient analyses, fulfilling the needs of the emerging biotechnology industry for microscale and massively parallel assays. The two primary approaches in miniaturizing electrophoresis dimensions are the capillary and microslab formats. This latter one evolved towards ultrathin-layer gel electrophoresis which is, except from the thickness of the separation platform, slightly in the upper side of the scale, resulting in considerably easier handling. Ultrathin-layer gel electrophoresis combines the advantages of conventional slab-gel electrophoresis (multilane format) and capillary gel electrophoresis (rapid, high-efficiency separations). It is readily automated, automatic versions of it have been extensively used for large-scale DNA sequencing in the Human Genome Project and more recently became popular in high throughput DNA fragment analysis. Ultrathin-layer techniques are the first step towards the wider use of electrophoresis microchips in perfecting a user-friendly interface between the user and the microdevice.

Animals↗
PubMed1999

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.

DNA Mutational Analysis↗
PubMed1999

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.

DNA↗
PubMed1999

Sample stacking during membrane-mediated loading in automated DNA sequencing.

Microporous membrane-mediated loading is a novel and efficient sample injection technique for ultrathin slab gel electrophoresis-based automated DNA sequence analysis. The sequencing reaction mixture is spotted directly onto the tabs of the membrane loader, which is then inserted to close proximity of the straight edge of the separation gel. The use of a higher viscosity (> 60 cSt), low ionic strength (conductivity < 320 microS) well solution with a pH of more than one unit lower that of the separation and gel buffer system makes possible efficient stacking of the DNA sequencing fragments at the interface of the gel matrix. When the injection/separation process is initiated by the application of the electric field, a high local electric potential drop is formed across the low ionic strength well solution zone. This results in very fast migration of the DNA sequencing fragments toward the interface of the separation gel, where they stack up against the higher conductivity sieving matrix. During this stacking process, primarily the local pH mediates the actual mobility of the buffer co-ions (borate), forming the leading and terminating zones in the well solution and separation gel, respectively.

Base Sequence↗
PubMed1999

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments.

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2 X or 4 X 48 base pair repeats) of the dopamine D4 receptor gene (D4DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.

DNA↗
PubMed1999

Human dopamine D4 receptor allele genotyping by ultrathin agarose gel electrophoresis with To-Pro-3 complexation.

Growing evidence shows the correlation between the allelic type of the dopamine D4 receptor and the human novelty-seeking personality trait. A sensitive, ultrathin agarose gel electrophoresis-based, high-throughput screening method was developed for genotyping the dopamine D4 receptor (D4DR) exon III 48 base pair repeat polymorphism. The efficiency of the method was increased by reamplification nested polymerase chain reaction (PCR) - of the 48 base pair repeat containing the PCR product with internal primers. The nested PCR fragments were analyzed by ultrathin layer agarose gel electrophoresis with an automated real-time laser-induced fluorescent detection system. Noncovalent affinity complexation was accomplished during the separation process by the addition of a very low concentration of intercalation dye, To-Pro-3 (2 nM) to the gel buffer system. This resulted in instant fluorescent labeling of the migrating PCR fragments. This method can readily facilitate genetic association studies between dopamine receptor genotypes and some human behavioral and neuropsychiatric disorders.

Alleles↗
PubMed1999

Efficacy of inhaled steroids (beclomethasone dipropionate) for treatment of mild to moderately severe asthma in the emergency department: a randomized clinical trial.

STUDY OBJECTIVE: To examine the efficacy of an inhaled steroid, when added to a standard regimen of beta-agonist therapy, in the treatment of patients with mild to moderately severe asthma in the emergency department. METHODS: A convenience sample of adult patients with asthma (FEV1 % predicted 40% to 69%) presenting to the ED was randomly assigned in a double-blind fashion into 2 treatment groups. The first group received 2.5 mg nebulized salbutamol plus 1 mg (4 puffs) of beclomethasone dipropionate (BDP) at baseline, 30 minutes, and at 1, 2, and 4 hours, delivered by a metered-dose inhaler (MDI) attached to a spacer device (Vent-AH-aler, Glaxo). The second group was given the same salbutamol regimen plus MDI placebo through the Vent-AH-aler. The primary endpoint was improvement in FEV1 %predicted at 6 hours. RESULTS: Of 54 patients enrolled, 28 were assigned to the BDP group and 26 to the placebo group. Spirometry improved significantly in both groups over the 6 hours compared with baseline (ANOVA, P <.001). At 6 hours, the mean absolute improvement in FEV1 % predicted for BDP was 18% versus 17% for placebo (95% confidence interval for the absolute difference of 1% [-8% to 10%]). The proportion of patients in the BDP group who were hospitalized was 7% compared with 19% for patients in the placebo group (95% confidence interval for the difference of 12% [-6%, 30%]). CONCLUSION: In this group of patients with mild to moderately severe asthma, 5 mg BDP delivered by MDI during the initial 4 hours of an emergency visit was of no added benefit over standard therapy, as measured by improvement in FEV1 % predicted at 6 hours. However, a trend toward a difference in admission favoring BDP was observed. [Afilalo M, Guttman A, Colacone A, Dankoff J, Tselios C, Stern E, Wolkove N, Kreisman H: Efficacy of inhaled steroids (beclomethasone dipropionate) for treatment of mild to moderately severe asthma in the emergency department: A randomized clinical trial.

Administration, Inhalation↗