Optimizing the immobilization of single-stranded DNA onto glass beads.
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 microg/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 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.
Department of Nutrition and Food Sciences, Center for Microbial Detection and Physiology, Utah State University, Logan, UT 84322-8700, USA.
M K
MK
Walsh
X
X
Wang
B C
BC
Weimer
D004277:DNA, Single-Stranded / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide; D005898:Glass; D007202:Indicators and Reagents; D008722:Methods; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013499:Surface Properties; D013861:Thiocyanates
D004277:DNA, Single-Stranded; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D013861:Thiocyanates; C032582:bitoscanate; D005022:Ethyldimethylaminopropyl Carbodiimide