Simple DNA probe assays based on particle agglutination.

I describe the use of latex agglutination as a simple readout for DNA hybridization assays. Latex agglutina-tion is a well-known readout for antibody-based diagnos-tics (1, 2) and uses " sandwich " assay chemistry for which analogous DNA assays are well known (3, 4). Latex agglutination is extremely simple and chemically robust, and latex particles have been used as a label in DNA hybridization (5). Here I show that simple agglutination of particles is an efficient measure of DNA hybridization, one that is insensitive to the presence of proteases, detergents , and solvents. Cloned m13 DNAs from the cytochrome f gene of Phormidium laminosum were kindly provided by Chris Howe (Department of Biochemistry, Cambridge, UK) (6). m13 DNA was prepared essentially as described by Maniatis et al. (7). Polystyrene microparticles (5.6-␮m polyvinylstyrene particles, density 1.060 kg/L; 4.5-␮m polystyrene/divinylbenzene particles, density 1.100 kg/L) were obtained from Bangs Laboratories. Nitrocel-lulose filters were obtained from Schleicher & Schuell. All other reagents were obtained from Sigma Chemicals. DNA was cross-linked onto polystyrene, using 1-ethyl-3,3-(3-dimethylaminopropyl)-carbodiimide (EDAC), according to the methods of Nikiforov et al. (8) and Vary (9). Two milligrams of DNA was incubated overnight with 2 mg of EDAC and 25 mg of beads in a total volume of 330 ␮L of water at 18 °C. Another 10 ␮L of 0.1 g/mL EDAC was added, and the incubation was continued for 1 h. Coupled beads were washed three times in 0.1ϫ standard saline citrate (SSC; 1ϫ SSC ϭ 8.765 g of NaCl, 4.1 g of disodium citrate per liter of water), stored at 4 °C. Particles with a reduced surface density of DNA were prepared by reacting 2 mg of DNA with 2 mg of EDAC and 25 mg of beads in a total volume of 330 ␮L of water at 18 °C for 1 h; then the particles were washed. DNA was immobilized onto polystyrene plate surfaces using exactly the same protocol. All hybridizations were carried out in 5ϫ SSC, 0.1% sodium dodecyl sulfate (SDS) at 60 °C in an orbital shaker. A unified agglutination index (AI) was calculated from photomicrographs as follows: AI ϭ ͑10.S ϩ 2.T ϩ 5.M ϩ 100.L͒ ͑100.S ϩ 5.T ϩ M ϩ L͒ These constants were selected to reflect the growth kinet-ics of particle aggregates (10). The density of particle aggregates was measured by centrifugation through a sucrose density gradient (1–12% sucrose in 5ϫ SSC, 0.01% SDS) in …