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.
Walsh
M K
MK
Department of Nutrition and Food Sciences, Center for Microbial Detection and Physiology, Utah State University, Logan, UT 84322-8700, USA.
Wang
X
X
Weimer
B C
BC
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