Analysis of DNA coverage using enzymatic cleavage of fluorescent labels

Abstract

In this study, a method to determine the degree of DNA coverage on gold surface using a fluorescent dye and Deoxyribonuclease I (DNase I) was developed. Gold surfaces were first functionalized with fluorescent dye labeled DNA, which was followed by DNase T digestion. The number of released fluorescent dyes represented the quantity of DNA immobilized. The use of DNase I to enzymatically cleave the fluorescent dye decreased the time needed for quantification to 1 h. In this way, the effect of a back-filler, DNA concentration, incubation time and spacer on DNA immobilization via gold-thiol chemistry was examined. The concentration of oligonucleotides and the ionic environment were the major factors determining DNA density. We also found that the length of DNA molecule influenced the rate of immobilization on Au. In addition, mixed self-assembled monolayer (mixed-SAM) with various composition and the the effect on DNA density was investigated.