Hybridization and Enzymatic Extension of Au Nanoparticle-Bound Oligonucleotides
| Autor: | Surabhi Raina, Nina V. Fedoroff, Glenn P. Goodrich, Sheila R. Nicewarner Peña, Christine D. Keating |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
biology
DNA polymerase Chemistry Oligonucleotide Oligonucleotides Nucleic Acid Hybridization DNA DNA-Directed DNA Polymerase General Chemistry Biochemistry Combinatorial chemistry Catalysis Primer extension Nucleic acid thermodynamics Colloid and Surface Chemistry biology.protein Nanotechnology Gold Multiplex ligation-dependent probe amplification Primase Particle Size Primer (molecular biology) Linker DNA Primers |
| Zdroj: | Journal of the American Chemical Society. 124:7314-7323 |
| ISSN: | 1520-5126 0002-7863 |
| Popis: | We have investigated the impact of steric effects on the hybridization and enzymatic extension of oligonucleotides bound to 12-nm colloidal Au particles. In these experiments, a nanoparticle-bound 12-mer sequence is hybridized either to its solution phase 12-mer complement or to an 88-mer template sequence. The particle-bound oligonucleotide serves as a primer for enzymatic extension reactions, in which covalent incorporation of nucleotides to form the complement of the template is achieved by the action of DNA polymerase. Primers were attached via-C(6)H(12)SH, -C(12)H(24)SH, and -TTACAATC(6)H(12)SH linkers attached at the 5' end. Primer coverage on the nanoparticles was varied by dilution with (5')HSC(6)H(12)AAA AAA(3'). Hybridization efficiencies were determined as a function of linker length, primer coverage, complement length (12-mer vs 88-mer), and primer:complement concentration ratio. In all cases, hybridization for the 88-mer was less efficient than for the 12-mer. Low primer surface coverage, greater particle-primer separation, and higher primer:complement ratios led to optimal hybridization. Hybridization efficiencies as high as 98% and 75% were observed for the 12-mer and 88-mer, respectively. Enzymatic extension of particle-bound primers was observed under all conditions tested; however, the efficiency of the reaction was strongly affected by linker length and primer coverage. Extension of primers attached by the longest linker was as efficient as the solution-phase reaction. |
| Databáze: | OpenAIRE |
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