Optimizing the immobilization of single-stranded DNA onto glass beads

Abstract

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo 1, onto glass beads via a 5′-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo 1 to aminopropyl glass beads. The influence of coupling buffer, oligo 1 concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82–89%. EDC concentrations greater than 50 mM and oligo 1 concentrations of 3 μg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo 2, was able to hybridize to the immobilized oligo 1 with a 58% efficiency. This oligonucleotide was subsequently released at 70°C. The relationship between the surface density of oligo 1 and the hybridization efficiency of the complementary oligonucleotide is described.