Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles

Citation
Jr. Taylor et al., Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles, ANALYT CHEM, 72(9), 2000, pp. 1979-1986
Citations number
71
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Chemistry & Analysis","Spectroscopy /Instrumentation/Analytical Sciences
Journal title
ANALYTICAL CHEMISTRY
ISSN journal
0003-2700 → ACNP
Volume
72
Issue
9
Year of publication
2000
Pages
1979 - 1986
Database
ISI
SICI code
0003-2700(20000501)72:9<1979:PSSOSD>2.0.ZU;2-0
Abstract
Nanometer-sized fluorescent particles (latex nanobeads) have been covalentl y linked to DNA binding proteins to probe specific sequences on stretched s ingle DNA molecules. In comparison with single organic fluorophores, these nanoparticle probes are brighter, are more stable against photobleaching, a nd do not suffer from intermittent on/off light emission (blinking). Specif ically, we demonstrate that the site-specific restriction enzyme EcoRI can be conjugated to 20-mn fluorescent nanoparticles and that the resulting nan oconjugates display DNA binding and cleavage activities of the native enzym e. In the absence of cofactor magnesium ions, the EcoRI conjugates bind to specific sequences on double-stranded DNA but do not initiate enzymatic cut ting. For single DNA molecules that are stretched and immobilized on a soli d surface, nanoparticles bound at specific sites can be directly visualized by multicolor fluorescence microscopy. Direct observation of site-specific probes on single DNA molecules opens new possibilities in optical gene map ping and in the fundamental study of DNA-protein interactions.