DNA hybridization on microparticles: Determining capture-probe density and equilibrium dissociation constants

Priscilla Wilkins Stevens, Michael R. Henry, David M. Kelso

Research output: Contribution to journalArticlepeer-review

64 Scopus citations

Abstract

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-selfcomplementary sequences that can be used to calculate the equilibrium dissociation constant (Kd) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.

Original languageEnglish (US)
Pages (from-to)1719-1727
Number of pages9
JournalNucleic acids research
Volume27
Issue number7
DOIs
StatePublished - Apr 1 1999

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'DNA hybridization on microparticles: Determining capture-probe density and equilibrium dissociation constants'. Together they form a unique fingerprint.

Cite this