Employing Microspheres for in Situ Hybridization Studies

V. Tohver Milam, N. Eze
Georgia Institute of Technology, US

Keywords: microsopheres, flow cytometry, LNA, DNA, hybridization rate constants

Summary:

Oligonucleotide-functionalized microspheres serve as convenient substrates in recognition-based assembly and nucleic acid detection schemes. Duplex densities can be conveniently measured on microspheres using flow cytometry; however, posthybridization wash steps are typically employed to remove any excess or weakly bound target species from the suspension prior to analysis. Here, we explore an alternative experimental approach in which we intentionally skip post-hybridization wash steps in order to directly assess primary duplex formation as it occurs between single-stranded probe-functionalized microspheres and soluble targets. These studies provide insight into the comparative hybridization activity of natural and modified oligonucleotides on material surfaces.