|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
50. A Quantitative Analytical Tool for Improving DNA-Based Diagnostic Arrays
Tom J. Whitaker
Sequence analysis using hybridization on ODN arrays is particularly well suited for genetic diagnostics, sequencing cDNAs, and partial sequencing of clones to allow mapping. In spite of this, quality control issues have hampered the full acceptance of these arrays, which are often called "gene chips". A high dynamic range, quantitative measurement method is needed to study parameters that increase efficiency and new methods of array production. With such a tool, systematic studies of hybridization strategies could be undertaken which would almost certainly lead to improved efficiencies and lower array manufacturing costs. Although fluorescence detection has been adequate for analysis of the hybridized chips, it does not have the spatial resolution or dynamic range to image the surface density of small (e.g. 20m spot size) bound probe ODNs or to perform hybridization kinetics studies.
We have just begun a new project to develop and utilize a high-resolution, quantitative method to analyze ODNs on an array. The technique involves detection of tin-labeled ODNs by sputtering them from the surface with an energetic ion beam, selectively ionizing the resulting tin atoms with wavelength-tunable lasers, and analyzing the ions with time-of-flight mass spectrometry. We have previously shown that this sputter-initiated resonance ionization microprobe (SIRIMP) technique can have sub-mm resolution and is highly quantitative in measuring a wide range of concentrations of elements in semiconductors. We have also shown that SIRIMP can detect DNA fragments labeled with stable isotopes 1,2. We now plan to apply SIRIMP to measurements of tin-labeled ODNs immobilized on a surface and to tin-labeled ODNs synthesized in situ on the surface. The initial phases will be used to develop and demonstrate the technique and calibration procedures. In later stages, we will work closely with Affymetrix to analyze and image in situ arrays with very small (20m) features to determine the homogeneity of binding. Additionally, hybridization experiments will be performed with two different stable isotopes of tin labeling the probe and target ODNs to determine the correlation between the surface density of the immobilized probes and hybridized targets.
The research reported here was funded, in whole or in part, by DOE grant #DE-FG02-98ER82536. Such support does not constitute an endorsement by DOE of the views expressed in this abstract.
1H.F. Arlinghaus, M.N. Kwoka, X.-Q. Guo and K.B. Jacobson, Analytical Chemistry 69, 1510 (1997).
2H.F. Arlinghaus, M.N. Kwoka, and K.B. Jacobson, Analytical Chemistry 69, 3747 (1997).
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