DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

40. Hybridization Detection

Tom J. Whitaker and Kenneth F. Willey

Atom Sciences, Inc., Oak Ridge, TN 37830

whitaker@atom-sci.com

Two projects aimed at developing new techniques that measure DNA hybridization to oligonucleotide (ODN) probes on DNA chips will be described. Although these techniques have similar goals, they differ widely in cost, complexity, sensitivity, and application. One method uses laser-based mass spectrometry analysis of stable isotopes of Sn atoms sputtered from Sn-labeled DNA target molecules. A 10-kV ion beam is used to sputter the sample from the surface and a wavelength-tunable laser efficiently and selectively ionizes the neutral Sn atoms for time-of-flight mass spectral analysis. The extreme sensitivity of this laser ionization technique has previously enabled high spatial resolution measurements of trace impurities in a variety of substrates. The technique also facilitates quantitative measurements by avoiding the effects of variation in secondary ion yield, a problem that plagues SIMS (secondary ion mass spectrometry) analyses. We are currently working with samples supplied by Affymetrix, Inc. to explore application of the technique for quality control of in-situ formation of ODN probes and DNA hybridization.

The second technique uses an inexpensive electronic detection method to determine if hybridization has occurred at a specific probe site. The low cost and small dimensions of this device make it ideal for point-of-care applications. The method takes advantage of the fact that an ODN probe can form an insulating self-assembled monolayer between a gold surface (on which the dielectric is attached) and a conducting liquid. Because of the extremely thin dielectric, the resulting capacitor has a very high specific capacitance. Calculations indicate that the change in effective dielectric thickness caused by the binding of a fraction of a monolayer of DNA to the ODN probe layer will produce a significant, and easily measurable, change in capacitance. Results of initial experiments aimed at verifying this concept will be provided and future experiments to enhance the capacitance change will be described.