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.
Ronai
Z
Z
Novartis Agricultural Discovery Institute, La Jolla, CA 92121, USA.
Guttman
A
A
Nemoda
Z
Z
Staub
M
M
Kalasz
H
H
Sasvari-Szekely
M
M
D001331:Automation; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005838:Genotype; D006801:Humans; D009061:Mouth Mucosa / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D017448:Receptors, Dopamine D2 / Q000235:genetics*; D050638:Receptors, Dopamine D4; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
C493599:DRD4 protein, human; D017448:Receptors, Dopamine D2; D050638:Receptors, Dopamine D4; D004247:DNA