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Technical Briefs |
a e-mail william{at}wbains.u-net.com
I describe the use of latex agglutination as a simple readout for DNA hybridization assays. Latex agglutination is a well-known readout for antibody-based diagnostics (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-µm polyvinylstyrene particles, density 1.060 kg/L; 4.5-µm polystyrene/divinylbenzene particles, density 1.100 kg/L) were obtained from Bangs Laboratories. Nitrocellulose 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 µL of water at 18 °C. Another 10 µL of 0.1 g/mL EDAC was added, and the incubation was continued for 1 h. Coupled beads were washed three times in 0.1x standard saline citrate (SSC; 1x SSC = 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 µL 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 5x 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:
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The density of particle aggregates was measured by centrifugation
through a sucrose density gradient (1–12% sucrose in 5x SSC, 0.01%
SDS) in 5-ml polypropylene tubes at 20 °C in a Sorvall (DuPont)
OTD65B centrifuge equipped with a Sorvall AH627 swing-out rotor.
Centrifugation times and speeds were not critical, providing that
t > 6.4 x
10 revolutions/s (e.g., 8000 rpm for 10 min).
The results of counting agglutinated particles with a microscope are
given in Fig. 1
. This measure is simple and allows sensitive, quantitative
measurement of the degree of agglutination achieved. Many of these
experiments also showed "clumping" of the particles, which was
visible to the unaided eye. Particles that were shaken with DNA that
hybridizes to both particles showed clear clumping into large
aggregates. Particles shaken with DNA that does not hybridize to both
particles or shaken without DNA showed only slight agglutination.
Agglutination was dependent on the amount of DNA loaded onto the
surface of the particles, as well as the amount in solution.
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Fig. 1
also illustrates that performing hybridization at lower
temperatures results in a slightly lower AI. The rate of agglutination
of particles is limited by particle collision kinetics (10),
which are not markedly affected by temperature (for these large
particles); therefore, the decrease in agglutination with temperature
is not as extreme as would be expected from considerations of DNA
hybridization kinetics (11). Decreasing temperature also
increases nonspecific binding slightly. Most nonspecific binding is
caused by static charging of the particles, as illustrated by the
nonzero agglutination at zero DNA concentration in Fig. 1
. Increasing
reaction time increases the cumulative particle collision probability
but also mechanically disrupts large aggregates; therefore, it only
increases assay sensitivity when the target DNA concentration is low.
At high concentrations, the agglutination is essentially complete in 25
min.
I also measured agglutination by detecting the binding of DNA-coated particles to the base of a DNA-coated microtiter plate in the presence of complementary target DNA, an assay similar to that described by Vener et al. (5), and by measuring the density of aggregates that result when DNA-coated particles of two different densities hybridize to a target. The sucrose gradient separated the two original particles (high and low densities) from clusters of particles (intermediate density). The extent of hybridization is measured by the fraction of beads that have moved from the top and bottom bands into the central region of the tube.
The above experiments are summarized in Table 1
. Both methods show agglutination caused by target DNA at 1
x 10-8 g/L in a 30-µL reaction volume,
corresponding to ~1.5 x 10 genomes of target. This
result is sensitive to the presence of DNase but not protease (Table 1
); it is resistant to the presence of SDS (which was included in all
hybridizations to reduce nonspecific particle agglutination), nonionic
detergents, and 5% ethanol (data not shown).
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This report shows that particle agglutination can be used as a readout technology for DNA hybridization. This is a distinct application of DNA immobilization from other applications reported previously, such as the immobilization of DNA to beads for kinetic studies (12), for posthybridization labeling (5) or chromatographic separations (13)(14), or to 96-well microtiter plates (15) to aid PCR. In this study, as in latex-based immunoassays, the microparticles are both the solid support on which hybridization is carried out and the readout technology by which we determine that hybridization has occurred. The model reactions shown here provide clear indications of the presence of target DNA at concentrations of 100 pg (0.5 pmol) in times comparable with typical PCRs (16). Test sensitivity is dependent on hybridization time, temperature, and probe density on the particles. Surprisingly, this simple readout technology can give greater sensitivity than a comparable sandwich hybridization using radiolabeled m13 probes (4).
Particle agglutination tests are simple to make and to operate. DNA probe-based tests using particle agglutination are thus attractive to smaller laboratories and less highly automated environments. Surveys of existing applications of DNA probe-based tests show that the sensitivity achieved in these model experiments is sufficient for a substantial proportion of medical diagnostic applications of DNA probe-based tests (17).
Acknowledgments
My thanks to Mary Meza (Bangs Labs) and Dan Brown (MRC Cambridge) for help with coupling chemistry, to Chris Howe for providing m13 clones, and to Nick Ashley and Lyn Scott for encouragement and comments .
Footnotes
Merlin Ventures, 101 Beechwood Ave., Melbourn, Royston, Herts SG8 6BW, UK
References
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