Control of Immobilization and Hybridization on DNA Chips by Fluorescence Spectroscopy

Abstract

A new methodology for the analysis of DNA polymorphisms has been developed using specific oligonucleotide strand arrays bound to a solid silicon support recovered by a thin layer of silica. Arrays of directly synthesized oligonucleotides covalently fixed on Si/SiO₂ wafers have been designed at the macroscopic scale. Using suitable nucleotide-labeled units, the fluorescence emission technique has been used as an experimental control of the molecular network bound to the support and as a method for analyzing the hybridizing abilities of the corresponding oligonucleotide array. Fluorescein has allowed us to control the molecular density of the DNA strand resulting from a complete synthetic growing process. A specific protocol using both complementary and noncomplementary units labeled with two probes, Cy3 and Cy5, was used to distinguish clearly nucleotide units fixed on the array either as hybridized sequences or by the unavoidable adsorption process. The present performance of this fluorescence detection procedure will now be used with a scanning fluorescence device to perform the analysis at the microscopic scale.