In situ synthesis of oligonucleotide arrays by using surface tension

This work describes the in situ synthesis of oligonucleotide arrays on glas s surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photol ithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to th e glass through a siloxane linkage. Each feature is bounded by a perfluoros ilanated 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 cont act angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secon dary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-ba sed oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates. an average synthesise efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%; Step yields measured for the individual amidite bases showed efficien cies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second meth od for determining the amidite coupling efficiencies was by capillary elect rophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), a nd dC (40mer) were synthesized on an NH4OH 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 cal culated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).