Optimizing the immobilization of single-stranded DNA onto glass beads

M. K. Walsh, X. Wang, Bart C Weimer

Research output: Contribution to journalArticle

114 Scopus citations

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), oligo1, 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 oligo1 to aminopropyl glass beads. The influence of coupling buffer, oligo1 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 oligo1 concentrations of 3 μg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo2, was able to hybridize to the immobilized oligo1 with a 58% efficiency. This oligonucleotide was subsequently released at 70°C. The relationship between the surface density of oligo1 and the hybridization efficiency of the complementary oligonucleotide is described.

Original languageEnglish (US)
Pages (from-to)221-231
Number of pages11
JournalJournal of Biochemical and Biophysical Methods
Volume47
Issue number3
DOIs
StatePublished - Feb 26 2001
Externally publishedYes

Keywords

  • Hybridization
  • Immobilization
  • Oligonucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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