Use of modified glasses for a microarray-based diagnosis of BRCA mutations

Abstract

In this paper, we describe a microchip-based BRCA diagnosis using slide glasses modified with covalent functional groups. First, we activated pre-synthesized aminosilylated glasses with isothiocyanate (NCS) and epoxide functional groups, respectively. The two kinds of modified glasses were then evaluated for their immobilization efficiency using capture probe oligonucleotides labeled with biotin at the 3' end, which can directly generate a fluorescence signal using the staining of the immobilized probes with streptavidin-Cy3 instead of their hybridization. The immobilization efficiencies of both the epoxide- and NCS-activated glasses were good enough to be useful as a substrate for an oligonucleotide microchip. Next, we examined their clinical utility for a reliable diagnosis of selected BRCA mutations through the hybridization of Cy3-labeled target DNA. All of the genotypes on the BRCA mutation sites were successfully identified on the two types of modified glass chips, providing reliable discrimination ratios (Qpm) between perfectly matched and mismatched probes of higher than 3.0 for all the mutation sites. This work demonstrates that both epoxide- and NCS-activated glasses can be good candidates for the preparation of a DNA chip for the diagnosis of human genetic mutations such as those found in BPCA genes.