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

Szöke, Melinda; Sasvári-Székely, Mária; Barta, Csaba; Guttman, András

doi:10.1002/(sici)1522-2683(19990301)20:3<497::aid-elps497>3.0.co;2-s

Electrophoresis

1999

DOI: 10.1002/(sici)1522-2683(19990301)20:3<497::aid-elps497>3.0.co;2-s

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

Melinda Szöke

Mária Sasvári-Székely

Csaba Barta

et al.

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Cited by 8 publications

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“…Since our miniaturized FIGE device is small enough to be placed under a fluorescence microscope, the separation process can be monitored during the course of the run. Mini-slab gels have been previously introduced for continuous field DNA separations [23][24][25][26][27][28][29][30][31], but have not been widely investigated for PFGE. Our apparatus ( Fig.…”

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Investigating DNA migration in pulsed fields using a miniaturized FIGE system

Chen

Ugaz

2008

Electrophoresis

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Pulsed field gel electrophoresis (PFGE) is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here we present a simple miniaturized field inversion gel electrophoresis (FIGE) apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 hours using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N −0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.Keywords pulsed field gel electrophoresis; field inversion gel electrophoresis; DNA As DNA fragment lengths approach tens of kilobases, a point is reached where individual molecules become much larger than the average pore size of the gel matrix. In order to sustain electrophoretic migration in this regime, the DNA molecules must adopt extended conformations preferentially aligned in the electric field direction. But this mode of migration is accompanied by a drastic reduction (and ultimately saturation) in the size dependence of electrophoretic mobility, making separation of long DNA fragments under continuous electric fields extremely challenging if not impossible. One way to overcome these limitations involves introducing a dynamic reorientation process by periodically changing the direction and magnitude of the applied electric field. When the electric field direction is switched, smaller fragments are able to reorient more quickly than larger molecules and thus migrate faster. The size dependent nature of this reorientation process restores the ability to distinguish different sized fragments based on their electrophoretic mobility. These pulsed field gel electrophoresis (PFGE) techniques have greatly extended the range of DNA fragment sizes that can be separated, even up to tens of Mb [1,2]. PFGE methods are important in a variety of applications including analysis of microbial genomes [3][4][5] [7][8][9][10][11]. But despite the instrumental role of PFGE techniques in broadening the range of DNA fragment sizes that can be effectively separated, some important drawbacks remain. First, PFGE can be extremely time consuming, with run times generally ranging in the tens of hours. A major reason for this is that the extent to which the electric field can be increased to drive faster DNA migration is limited by the ...

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confidence: 99%

Investigating DNA migration in pulsed fields using a miniaturized FIGE system

Chen

Ugaz

2008

Electrophoresis

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

et al. 2000

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

Rónai

Guttman

Nemoda³

et al. 2000

Electrophoresis

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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.

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

Cited by 8 publications

References 18 publications

Investigating DNA migration in pulsed fields using a miniaturized FIGE system

Investigating DNA migration in pulsed fields using a miniaturized FIGE system

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

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

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