Probing Specific Sequences on Single DNA Molecules with Bioconjugated Fluorescent Nanoparticles

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• 作者：Jason R. Taylor ; Michelle M. Fang ; and Shuming Nie
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：May 1, 2000
• 年：2000
• 卷：72
• 期：9
• 页码：1979 - 1986
• 全文大小：1224K
• 年卷期：v.72,no.9(May 1, 2000)
• ISSN：1520-6882

文摘

Nanometer-sized fluorescent particles (latex nanobeads)have been covalently linked to DNA binding proteins toprobe specific sequences on stretched single DNA molecules. In comparison with single organic fluorophores,these nanoparticle probes are brighter, are more stableagainst photobleaching, and do not suffer from intermittent on/off light emission (blinking). Specifically, wedemonstrate that the site-specific restriction enzymeEcoRI can be conjugated to 20-nm fluorescent nanoparticles and that the resulting nanoconjugates display DNAbinding and cleavage activities of the native enzyme. Inthe absence of cofactor magnesium ions, the EcoRIconjugates bind to specific sequences on double-strandedDNA but do not initiate enzymatic cutting. For single DNAmolecules that are stretched and immobilized on a solidsurface, nanoparticles bound at specific sites can bedirectly visualized by multicolor fluorescence microscopy.Direct observation of site-specific probes on single DNAmolecules opens new possibilities in optical gene mappingand in the fundamental study of DNA-protein interactions.