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

Abstract

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.

Association of D4 dopamine receptor gene and serotonin transporter promoter polymorphisms with infants' response to novelty

“…32 5-HTTLPR genotypes were determined from the same DNA sample according to Heils et al 6 with some modifications (primers used: 5 0 GGC GTT GCC GCT CTG AAT GC 3 0 and 5 0 GGC GTT GCC GCT CTG AAT GC 3 0 ; thermocycling was initiated at 951C for 10 min to activate HotStar DNA polymerase (Qiagen) followed by 35 cycles of 1 min denaturation at 951C, 1 min of annealing at 651C and 1 min extension at 721C, and completed by 10 min of extension at 721C). In both VNTR polymorphisms dGTP was replaced with dITP in 50% in order to avoid allelic drop-out in heterozygotes.…”

“…In both VNTR polymorphisms dGTP was replaced with dITP in 50% in order to avoid allelic drop-out in heterozygotes. 32 DRD4 48 bp VNTR genotype and allele frequencies in the BIPS sample population were the same as previously reported, 21 and were similar to the frequencies recently determined in a larger Hungarian population, showing a good agreement with the Hardy-Weinberg equilibrium (P40.9). 33 Frequencies of the 14-and the 16-repeat 5-HTTLPR alleles in the BIPS sample were 0.394 and 0.606, respectively.…”

Dopamine D4 receptor (DRD4) gene polymorphism is associated with attachment disorganization in infants

“…33 Great care was taken to avoid pseudohomozygosity, using parallel determinations of different DNA template concentration and substituting 50% dGTP by dITP. 34 All the genotyping was done blind to the attachment status in the Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University.…”

Multicapillary Electrophoresis Analysis of Single-Nucleotide Sequence Variations in the Deoxycytidine Kinase Gene

“…Ultrathin-layer gel electrophoresis combines the multilane format of horizontal slab gel electrophoresis with the high resolving power of capillary-and microchip-based approaches (21 ). This technique has been used by our group for the investigation of several genetic sequence variations of neurotransmitter systems in psychogenetic association studies (22,23 ). Recently, multicapillary gel electrophoresis systems, such as the eGene HDA-GT12, were introduced to accommodate rapid DNA fragment analysis (24 ).…”