Polymer support for exonucleolytic sequencing

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene 'core' and a 'shell' of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 mum diameter. Applying M13 ssDNA in extremely high dilution (similar to 1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 mum diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand. (C) 2001 Published by Elsevier Science B.V.