In situ synthesis of oligonucleotide arrays by using surface tension

Abstract

This work describes the in situ synthesis of oligonucleotide arrays on glass surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photolithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to the glass through a siloxane linkage. Each feature is bounded by a perfluorosilanated surface. The differences in surface energies between the features and surrounding zones allow for chemical reactions to be readily localized within a defined site. The aminosilanation process was analyzed using contact angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secondary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-based oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates an average synthesis efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%. Step yields measured for the individual amidite bases showed efficiencies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second method for determining the amidite coupling efficiencies was by capillary electrophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), and dC (40mer) were synthesized on an NH(4)OH labile linkage. After cleavage, the products were analyzed by CE. Synthesis efficiencies were calculated by comparison of the full-length product peak with the failure peaks. The calculated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).