Human dopamine D-4 receptor allele genotyping by ultrathin agarose gel electrophoresis with To-Pro-3 complexation

Growing evidence shows the correlation between the allelic type of the dopa mine D-4 receptor and the human novelty-seeking personality trait. A sensit ive, ultrathin agarose gel electrophoresis-based, high-throughput screening method was developed for genotyping the dopamine D-4 receptor (D4DR) exon III 48 base pair repeat polymorphism. The efficiency of the method was incr eased by reamplification - nested polymerase chain reaction (PCR) - of the 48 base pair repeat containing the PCR product with internal primers. The n ested PCR fragments were analyzed by ultrathin layer agarose gel electropho resis with an automated real-time laser-induced fluorescent detection syste m. 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 fluoresc ent labeling of the migrating PCR fragments. This method can readily facili tate genetic association studies between dopamine receptor genotypes and so me human behavioral and neuropsychiatric disorders.