DNA-based single-molecule studies, nanoelectronics and nanocargos require a precise placement of DNA in an orientation-defined manner. Until now, there is a lack of orientation-defined alignment and immobilization of DNA over distances smaller than several micrometers. However, this can be realized by designing bifunctionalized DNA with thiol at one end and (3-aminopropyl) tri-ethoxy silane at the other end, which specifically binds to a gold and SiO₂ layer after and during alignment, respectively. The electrode assembly consists of platinum as the electrode material for applying the AC voltage and islands of gold and silicon dioxide fabricated at a distance of about 500-800 nm by electron-beam lithography. The orientation-defined alignment and covalent binding of pUC19 DNA to specific surfaces are carried out in frequency ranges of 50 Hz-1 kHz and 100 kHz-1 MHz and observed after metallization of DNA by palladium ions by field emission scanning electron microscopy (FESEM). The bifunctionalized 890 nm long DNA was effectively aligned and immobilized between a gap of 500 to 600 nm width.