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A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles

NCJ Number
252464
Date Published
2017
Length
11 pages
Author(s)
H X Yu
Agencies
NIJ-Sponsored
Grant Number(s)
2013-DN-BX-K032, 2015-R2-CX-0034
Annotation
This article presents research on the performance of DNA-modified particles used for applications in sensing, material science, and molecular biology.
Abstract
DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. The researchers developed a simple and broadly applicable exonuclease III digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Utilizing particle-conjugated, fluorophore-labeled probes that incorporates two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation via cleavage and digestion of surface-bound probe DNA via Exo apurinic endonucleolytic and exonudeolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. The researchers method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.
Date Created: July 20, 2021