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Biomedical subjects

A Guttman

Publications and source records attributed to A Guttman.

At least 19 recordsLinked to original sources
PubMed2005

The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region.

BACKGROUND: The human dopamine D4 receptor (DRD4) is a candidate gene of great interest in molecular studies of human personality and psychiatric disorders. This gene is unique in having an exceptionally high amount of polymorphic sites both in the coding and in the promoter region. RESULTS: We report the identification of a new 27 bp deletion starting 524 bp upstream of the initiation codon (27 bp del) of the dopamine D4 receptor (DRD4) gene, in the close vicinity of the -521C>T SNP. The presence of the 27 bp deletion leads to the misgenotyping of the -616C>G SNP by the Sau96 I RFLP method, thus the genotype determination of the mutation is of additional importance. The frequency of this novel sequence variation is considerably low (allele frequency is = 0.16%), as no homozygotes, and only 3 heterozygote carriers were found in a healthy, unrelated Caucasian sample (N = 955). CONCLUSION: Remarkably, the deleted region contains consensus sequences of binding sites for several known transcription factors, suggesting that the different alleles may affect the transcriptional regulation of the gene. A comparison of methods and results for the allelic variations of the DRD4 gene in various ethnic groups is also discussed, which has a high impact in psychiatric genetic studies.

Binding Sites↗
PubMed2004

A novel A/G SNP in the -615th position of the dopamine D4 receptor promoter region as a source of misgenotyping of the -616 C/G SNP.

The polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene containing several single nucleotide polymorphisms (SNPs) has recently become a focus of association studies in psychiatric genetics. Most SNP genotyping methods are based on the two-step procedure of restriction fragment length polymorphism (RFLP). An alternative technique is a single-step method of allele-specific amplification (ASA), previously introduced for genotyping the -521 C/T SNP of the DRD4 promoter region and applied here for the -616 C/G SNP. Parallel genotyping of individuals with the novel ASA method and the conventionally used Ava II RFLP showed a potential underestimation of the -616 GG genotype frequency by the conventional method. Sequencing the dubious samples clearly demonstrated a novel A/G SNP at the -615th position influencing the Ava II digestion and thus resulting in misgenotyping. To avoid this problem, we introduced the Sau96 I RFLP for the -616 C/G genotyping as this restriction enzyme is not sensitive for the -615 A/G sequence variation. Allele (-616 G = 0.48; -616 C = 0.52) and genotype (-616 GG = 0.25; -616 GC = 0.46; -616 CC = 0.29) frequencies were determined by both the novel ASA and the Sau96 I methods. The obtained genotype frequencies corresponded to the Hardy-Weinberg equilibrium in our healthy Caucasian sample (N = 534, P = 0.168). Using these methods, no association was found between the -616 C/G SNP and personality factors of Cloninger's temperament and character inventory (N = 153) in our population.

Gene Frequency↗
PubMed2004

Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene.

DNA-protein interaction in the 5' upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2 x 120 bp) or single copy (1 x 120 bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5' upstream region and suggest enhanced binding capacity of the duplicated form.

5' Flanking Region↗
PubMed2002

Noninvasive genotyping of dopamine receptor D4 (DRD4) using nanograms of DNA from substance-dependent patients.

A noninvasive DNA sampling method has been implemented collecting buccal mucosa cells by cotton wool swabs. An amount of 0.2 2 microg DNA per patient was obtained after the phenol-extraction procedure and 0.2 2 ng DNA template was sufficient for PCR amplification of the polymorphic 48 basepair repeat region of dopamine receptor D4 (DRD4) gene. PCR products were visualized during microfabricated electrophoretic separation by laser-induced fluorescent detection and automatic data registration. Initial data of genotyping drug-dependent subjects shows a relatively high ratio of heterozygotes, possessing either longer or shorter variants beside the common 4-repeat DRD4 allele.

Base Sequence↗
PubMed2001

Analysis of dopamine D4 receptor gene polymorphism using microchip electrophoresis.

A microfabricated electrophoresis device was used for rapid polymerase chain reaction product analysis in genotyping the dopamine D4 receptor gene (DRD4) 48 base pairs repeat polymorphism. An allelic ladder, prepared from homozygous individuals, was used as internal standard during the microchip electrophoresis based analysis. Comparison of this novel separation method with the conventional slab gel and previously reported ultra-thin-layer techniques confirmed the reliability of this new method. Genotyping of 332 healthy Hungarian individuals gave the following allele frequencies: two-repeat: 0.089; three-repeat: 0.026; four-repeat: 0.674; five-repeat: 0.011; six-repeat: 0.002; seven-repeat: 0.189; eight-repeat: 0.011. The genotype frequencies obtained showed no deviation from the Hardy-Weinberg equilibrium (p>0.903), further underlying the reliability of this new genotyping technique.

Base Sequence↗
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↗
PubMed2001

High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.

Alleles↗
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↗