Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechno logy and molecular biology applications. This paper compares different immo bilization chemistries to covalently link single-stranded DNA (20 base pair s), oligo(1), onto glass beads via a 5 ' -amino terminal end. Immobilizatio n methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimid e (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize o ligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo (1) concentration, and EDC concentration was also investigated. The one-ste p EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M M ES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89 %. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 m ug/g bead were required for effective immobilization. A complementary oligo nucleotide, oligo,, was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degr eesC. The relationship between the surface density of oligo(1) and the hybr idization efficiency of the complementary oligonucleotide is described. (C) 2001 Elsevier Science B.V. All rights reserved.