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Agarose Gel Electrophoresis

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András Guttman – One of the best experts on this subject based on the ideXlab platform.

  • High-throughput genotyping of factor V Leiden mutation by ultrathin-layer Agarose Gel Electrophoresis.
    Journal of chromatography. A, 1999
    Co-Authors: T Lengyel, M Sasvari-szekely, András Guttman

    Abstract:

    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.

  • Human dopamine D4 receptor allele genotyping by ultrathin Agarose Gel Electrophoresis with To-Pro-3 complexation.
    Electrophoresis, 1999
    Co-Authors: Melinda Szoke, Maria Sasvari-szekely, Csaba Barta, András Guttman

    Abstract:

    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.

  • Automated DNA fragment analysis by high performance ultrathin-layer Agarose Gel Electrophoresis
    , 1999
    Co-Authors: András Guttman

    Abstract:

    Ultrathin-layer Agarose Gel Electrophoresis combines the advantages of the multilane format of conventional slab and the high performance capabilities of capillary Gel Electrophoresis. This article describes the detection of separated sample components in real-time by using an integrated scanning laser-induced fluorescence-avalanche photodiode detection system. Ultrathin-layer Agarose Gel Electrophoresis is a novel and high performance separation technique that provides a simple platform for fast deoxyribonucleic acid fragment analysis with the advantage that the same Gel-filled cassette can be used simultaneously for a size range of 20-23130 base pairs. The author provides examples of rapid genotyping and high-throughput mutation analysis of polymerase chain reaction-generated fragments

Zdena Palkova – One of the best experts on this subject based on the ideXlab platform.

  • comparative analyses of saccharomyces cerevisiae rnas using agilent rna 6000 nano assay and Agarose Gel Electrophoresis
    Fems Yeast Research, 2003
    Co-Authors: Marketa řicicova, Zdena Palkova

    Abstract:

    Precise quantification and quality characterisation of isolated RNAs are prerequisites for their further exploitation in genome-wide microarrays, Northern blots, cDNA library preparation and others. Our data indicate that RNA analyses using Agilent RNA Nano Assay exhibit several advantages when compared with those performed on ethidium bromide-stained Agarose Gel Electrophoresis or on a spectrophotometer. The RNA Nano Assay makes it possible to estimate RNA concentrations in the range from 1000 ng μl−1 to 17 ng μl−1. The presence of impurities including traces of DNA within RNA samples does not influence the concentration measurements. Like Agarose Gel Electrophoresis, RNA Nano Assay allows to analyse RNAs dissolved in formamide and therefore protected against RNase action. Moreover, it allows a clearer distinction of partially degraded samples. The limitation of RNA Nano Assay is the impossibility to detect and to analyse double-stranded RNAs.

Jorge Bernardo Schvartzman – One of the best experts on this subject based on the ideXlab platform.

  • plasmid dna topology assayed by two dimensional Agarose Gel Electrophoresis
    Methods of Molecular Biology, 2013
    Co-Authors: Jorge Bernardo Schvartzman, Pablo E. Hernández, Maria Luisa Martinezrobles, Dora B Krimer

    Abstract:

    Two-dimensional (2D) Agarose Gel Electrophoresis is nowadays one of the best methods available to analyze DNA molecules with different masses and shapes. The possibility to use nicking enzymes and intercalating agents to change the twist of DNA during only one or in both runs, improves the capacity of 2D Gels to discern molecules that apparently may look alike. Here we present protocols where 2D Gels are used to understand the structure of DNA molecules and its dynamics in living cells. This knowledge is essential to comprehend how DNA topology affects and is affected by all the essential functions that DNA is involved in: replication, transcription, repair and recombination.

  • plasmid dna replication and topology as visualized by two dimensional Agarose Gel Electrophoresis
    Plasmid, 2010
    Co-Authors: Jorge Bernardo Schvartzman, Pablo E. Hernández, Maria Luisa Martinezrobles, Dora B Krimer

    Abstract:

    During the last 20 years, two-dimensional Agarose Gel Electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

  • Unidirectional replication as visualized by two-dimensional Agarose Gel Electrophoresis.
    Journal of Molecular Biology, 1991
    Co-Authors: Luis Martin-parras, Pablo E. Hernández, María Luisa Martínez-robles, Jorge Bernardo Schvartzman

    Abstract:

    Two-dimensional (2D) Agarose Gel Electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 μm plasmid. Neutral/Neutral (N/N) 2D Agarose Gel Electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D Gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D Agarose Gel Electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.