Genomic DNA hybridizes with the same rate constant on the QCM biosensor as in homogeneous solution

Abstract

Hybridization rates of sheared, genomic E. coli DNA in 0.14 M, pH 6.7 phosphate buffer at 65°C were determined by: (1) observing the rate of absorbance decrease at 260 nm due to self-hybridization in solution; and (2) measurement of the rate of mass increase caused by hybridization between DNA in solution and DNA photografted to polystyrene. The latter measurement was done using a quartz crystal microbalance (QCM). In both the spectrophotometric and QCM experiments the probe was identical to the target, as both were taken from the same sample of sheared E. coli DNA. In the QCM measurements, viscoelastic effects were made negligible by drying the biopolymer layer on the QCM's surface before taking the frequency readings. Our purpose was to explore the effect of immobilizing DNA on its hybridization rate constant. A second-order constant of 2.32±0.09×10 −6 ml μg −1 s −1, n=14, for hybridization in solution was obtained spectrophotometrically, while the QCM experiment gave a constant of 2.2±0.3×10 −6 ml μg −1 s −1, n=6. These values are not statistically different. The reaction half-lives for the spectrophotometric and QCM experiments were 6.5 h and 13 min, respectively. The shorter half-life on the QCM can be explained solely by the much greater reactant concentration in the QCM experiment. About 25% of the DNA was inactivated by the attachment reaction. After correcting for this, the surface-attached DNA hybridized with the same rate constant as DNA free in solution. Therefore, it is concluded that, in these specific experiments with genomic DNA, the immobilized regions must have been short compared to the length of the molecules. The data demonstrate the high hybridization rate obtainable when nucleic acids are hybridized in a thin-film, micro-volume reaction on a non-porous surface.