Capillary Electrophoresis Study on DNA-Protein Complex Formation in the Polymorphic 5 Upstream Region of the Dopamine D4 Receptor (DRD4) Gene

Z.      Ronai; A.      Guttman; G.      Keszler; M.      Sasvari-Szekely

Abstract

DNA-protein interaction in the 5 upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2x120 bp) or single copy (1x120bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5 upstream region and suggest enhanced binding capacity of the duplicated form.

Keywords: capillary electrophoresis, electrophoretic mobility shift assay, dopamine d4 receptor

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