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 μm diameter. Applying M13 ssDNA in extremely high dilution (∼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 μm 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.
Bibliographical noteFunding Information:
This work was supported from the German Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Grant no. 0310794-659) and from the German–Israeli Research Foundation (I-396-235.13/94). The studies in Israel were partially supported by Minerva (Otto Meyerhoff Center for the Study of Drug-Receptor Interactions).
- DNA carrier particles
- Dilution techniques
- Exonucleolytic sequencing
- Fluorescent labelling