Method of preparing an oligonucleotide array

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S287200, C536S022100, C536S023100

Reexamination Certificate

active

06790613

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is in the field biochips with arrays of porous polymer pads for analyzing biological samples.
2. Background of the Art
A biochip array is generally comprised of a solid substrate, a supporting matrix and a variety of biomolecule probes immobilized on the supporting matrix. When the biochip is exposed to a target that recognizes one of the immobilized probes, a binding event occurs, which results in a change of an electromagnetic signal such as fluorescence or impedance.
In addition to glass, porous polymer pads, especially polyacrylamide gel pads, have been used as supporting matrices for the attachment of bioactive specimens in a variety of biochip arrays (see U.S. Pat. Nos. 5,552,270; 5,616,478; 5,736,257; 5,741,700). Compared to glass, porous polymers have a much higher probe loading capacity owing to their porous three-dimensional nature.
In conventional polyacrylamide gel pads, the porosity is somewhat limited. The maximum reported pore size for polyacrylamide gel pads is only around 0.6 micron with 60% crossing linker (J. Biochem and Biophys. Methods, 4, 347 (1981). The gel pads with 5% crosslinker (acrylamide/bisacrylamide ratio 19:1) have an average pore size less than 0.1 micron. The pore size is generally believed to be too small for target DNA greater than a couple of hundred base pairs to diffuse into. Thus most binding events will only occur on the surface of the gel pads just as in the case of using glass as the supporting matrix. It is especially true for detection that requires some enzymatic reactions since enzyme molecules are generally much bigger in size and will not diffuse into small pores. To take advantage of the high loading capacity of porous polymer pads, it is necessary to have the ability to control the pore size distribution so that the target molecules can easily diffuse into the porous pads to be detected by the immobilized probes.
SUMMARY OF THE INVENTION
The invention involves the improvement of arrays of porous polymer pads on a solid support used in biological assays. The improvement involves freeze drying the porous polymer pads on solid support to increase the pore size. Thus it is an object of the invention to provide an array of macroporous polymer pads having specific binding agents such as DNA, RNA or polypeptides for use in biological assays. The invention provides for enhanced sensitivity by incorporating a larger amount of specific binding substance. Typically, an array of porous polymer pads on a solid support is frozen at liquid nitrogen temperatures and solvent is removed by sublimation of reduced pressure.


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Guschin, D., Yershov, G., Zaslavsky, A., Gemmel, A., Shick, V., Proudnikov, D., Arenkov, P., and Mirzabekov, A. 1997. “Manual manufacturing of oligonucleotide, DNA, and protein microchips.”Anal. Biochem.,vol. 250, No. 2, pp. 203-211.
Righetti, P., Brost, B., and Snyder, R. 1981. “On the limiting pore size of hydrophilic gels for electrophoresis and isoelectric focusing.”J. Biochem. Biophys. Methods,vol. 4, pp. 347-363.
Blank, Z., and Reimschuessel, A. 1974. “Structural studies of organic gels by SEM.”J. Mater. Sci.,vol. 9, pp. 1815-1822.
Rouchel, R., and Bager, M. 1975. “Scanning electron microscopic observations of polyacrylamide gels.”Anal. Biochem.,vol. 68, pp. 415-428.

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