Factors affecting the read-out of DNA suspension arrays

DNA suspension arrays provide the means for a high-throughput parallel analysis. However, hybridisation on solid supports has not been studied as extensively as in solution. The relationship between fluorescence read-out, hybridisation strength and selectivity, and solution and washing conditions of the array platform was investigated. Fluorescently labelled oligonucleotide targets were hybridised to oligonucleotide probes immobilised on avidin coated GMA beads. The DNA duplexes formed by the hybridisation were designed to contain a comprehensive set of single base mismatches centrally located in the double helix. The beads were subjected to a series of post-hybridisation washes with increasing stringency and duplex stability monitored by single bead fluorescence. The solution Tm for all duplexes was determined in a range of solvent conditions representing the different washing steps. It was found that the thermodynamic stability trend predicted by the solution Tms did not correlate the fluorescence read-out exhibited by the immobilised duplexes. Results suggested that the hybridisation discrimination achieved between complementary and mismatched duplexes was due to faster kinetics of dissociation of the mismatches under the washing conditions employed rather than discrimination based in thermodynamic stability. Solid phase single base extension and restriction endonuclease digestion of bead immobilised DNA duplexes were also explored. The performance of DNA polymerases and an endonuclease was investigated and compared in solution and on solid phase. Results showed that nucleotide incorporation is more effectively achieved in solution than on solid phase while solution and solid phase restriction digestions yielded similar results.