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 hu man traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorre ct genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase c hain reaction (PCR) products, resulting in the so-called allele dropout. In this paper we optimized the conditions for simultaneous amplification of s horter 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 genot yping, using very low DNA template concentrations and partial replacement o f 2'-deoxyguanosine-5'-triphosphate (dGTP) by 2'-deoxyinosine-5'-triphospha te (dITP). The optimized PCR method in combination with high throughput aut omated ultrathin-layer gel electrophoresis was suitable for rapid genotypin g from less than a nanogram DNA using noninvasive sampling (buccal epitheli al cells). All detected genotypes are presented, including such rear hetero zygotes as the 2 x and 8 x 48 bp repeats in the same sample, showing the re liability of our novel detection method of longer alleles in the presence o f shorter alleles.