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 microm diameter. Applying M13 ssDNA in extremely high dilution (approximately 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 microm 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.
Hinz
M
M
Sektion Polymere, University of Ulm, Albert-Einstein-Allee 11, D 89069 Ulm, Germany. micha_hinz@hotmail.com
Gura
S
S
Nitzan
B
B
Margel
S
S
Seliger
H
H
D001710:Biotin / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D008863:Microspheres; D011108:Polymers / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D019809:Streptavidin / Q000737:chemistry
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D011108:Polymers; D001710:Biotin; D019809:Streptavidin