H.F. Arlinghaus[1], Margaret N. Kwoka[1], K. Bruce Jacobson[2] and K. L. Beattie[3]
[1]Atom Sciences, Inc., [2]Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, TN and [3]Houston Advanced Research Center, Houston, TX
DNA hybridization chips were constructed by attaching 17-mer sequences from M13 DNA and T7 DNA to separate sites on a platinum film on a quartz surface. The platinum was applied in islands of ~5 mm diameter or it was spread uniformly over the surface. The DNA was attached to platinum by a newly devised chemistry. M13(-20) is a 17-mer, that is complimentary to a portion of the M13 DNA, and it was labeled with [118]Sn atoms[1]. Hybridization of the DNA on the chip with this 17-mer, and washing, occurred at 5° C.
The position of the tin label was identified with Sputter Initiated Resonance Ionization Spectroscopy (SIRIS), a technique that analyzes a fraction of a monolayer of the surface of any sample. In the case of platinum islands, the [118]Sn was found to be located on all of the complementary sites and none was present on the non-complimentary sites or the intermediary space. However, the tin was not well localized on the even film of platinum, indicating that a thin layer of tin had deposited on this surface. In contrast to [32]P or fluorescent labels, SIRIS detects the tin present in only a fraction of a monolayer of the surface. The amount of material that spreads out non-specifically probably would not be detectable by autoradiography or fluorescence but is apparent with SIRIS.
Successful use of SIRIS and tin-labeled DNA offers an alternative to fluorescence for analyzing hybridization chips. The signal-to-noise ratio of tinlabeled DNA was over 100 to 1 whereas the signal-to-noise of fluorescent labels is often less than 4 to 1, unless confocal microscopy is employed. SIRIS has the potential to analyze several hundred separate hybridization spots per second and thus offers a rapid means to obtain data from the hybridization chip. Furthermore, several isotopes of tin or other elements can be used on different DNA probes simultaneously to provide a rapid multiplex analysis of several DNAs at once.
*Research sponsored by the Laboratory Directed Research and Development Program of the Oak Ridge National Laboratory, managed for the U. S. Department of Energy by Lockheed Martin Energy Systems, Inc., under contract No DE-AC05-84OR21400 and two SBIR Phase I's to Atom Sciences, Inc. from NIH: 1-R43-CA66525-01 and 1-R43-MH52938-01.
[1] K.B. Jacobson and H.F. Arlinghaus, Anal. Chem. 64, 315A-328A (1992)