Graves, David J

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  • Publication
    Differential Adhesion of Microspheres Mediated by DNA Hybridization I: Experiment
    (2006-03-13) Zhang, Ying; Graves, David J; Milam, Valeria T; Hammer, Daniel A
    We have developed a novel method to study collective behavior of multiple hybridized DNA chains by measuring the adhesion of DNA-coated micron-scale beads under hydrodynamic flow. Beads coated with single-stranded DNA probes are linked to surfaces coated with single target strands through DNA hybridization, and hydrodynamic shear forces are used to discriminate between strongly and weakly bound beads. The adhesiveness of microspheres depends on the strength of interaction between DNA chains on the bead and substrate surfaces, which is a function of the degree of DNA chain overlap, the fidelity of the match between hybridizing pairs, and other factors that affect the hybridization energy, such as the salt concentration in the hybridization buffer. The force for bead detachment is linearly proportional to the degree of chain overlap. There is a detectable drop in adhesion strength when there is a single base mismatch in one of the hybridizing chains. The effect of single nucleotide mismatch was tested with two different strand chemistries, with mutations placed at several different locations. All mutations were detectable, but there was no comprehensive rule relating the drop in adhesive strength to the location of the defect. Since adhesiveness can be coupled to the strength of overlap, the method holds promise to be a novel methodology for oligonucleotide detection.
  • Publication
    Competitive Hybridization Kinetics Reveals Unexpected Behavior Patterns
    (2005-11-01) Hammer, Daniel A; Zhang, Ying; Graves, David J
    Although the kinetics of hybridization between a soluble polynucleotide and an immobilized complementary sequence have been studied by others, it is almost universally assumed that the interaction between each probe/target pair can be treated as a separate event. This simplifies the mathematics considerably, but it can give a false picture of the extent of hybridization that one achieves at equilibrium as well as the relative quantities of each hybridized pair during the approach to equilibrium. Here we solve the relevant kinetics equations simultaneously using Mathematica as a simulation language. Among the interesting results of this study are that, for certain circumstances, the relative ratio of incorrect to correct hybrids can change dramatically with time; that the relative abundances of two pairs are not what one would expect based on their equilibrium dissociation constants; that the volume of a wash solution after hybridization can have a large effect on results; and the fact that a short wash is typically better than a long one. We show that an optimum wash time exists for a given set of conditions. In addition, the ratio of soluble to insoluble (spotted) molecules can influence results substantially. Finally, the true levels of rare transcripts can be masked by the presence of highly abundant ones. Code is supplied to enable others to study conditions beyond those presented in this article.