Bio-molecular chip and manufacturing process thereof

Details Bio-molecular chip and manufacturing process thereof Bio-molecular chip and manufacturing process thereof

2001-03-06

2004-06-08

Whisenant, Ethan (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S287200, C435S288300, C530S300000, C530S387100, C536S023100, C536S024300

Reexamination Certificate

active

06746842

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to a bio-molecular microchip and a manufacturing process thereof. More particularly, the present invention is directed to a bio-molecular microchip comprising a substrate, and an amine group-containing bio-molecule that is immobilized onto a glycidyl moiety-containing polyacrylamide gel via covalent bonds formed between the glycidyl moiety of the polyacryl amide gel and the amine group of the bio-molecule by epoxy ring-opening reaction; a manufacturing process thereof.
BACKGROUND OF THE INVENTION
As is known, oligonucleotide hybridization technique wherein oligonucleotide or target DNA fragment is immobilized on a gel or solid surface, is applied in the various fields. Recently, these oligonucleotide hybridization technique has been applied to DNA sequencing method, and various kinds of research relating to them have been made(Barinaga, M. (1991) Science 253:1489; Cantor, C. R., Mirzabekov, A. and Southern, E. (1992) Genomics 13, 1378-1383; Southern, E. M., Maskos, U. and Elder, J. K. (1992) Genomics 13, 1008-1017; Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shai, N., Shen, N., Yang, R. and Fodor, S. P. A. (1995) Biotechniques 19: 442-447). Also, a number of methods for DNA immobilization on a gel or a solid surface have been developed. They are largely divided into two groups. One is to synthesize oligonucleotides directly on a glass surface, the other is to first synthesize oligonucleotides and immobilized this synthesized oligonucleotides on a solid surface or a gel surface.
For example, Southern et al. has disclosed a method for synthesizing oligonucleotides on a glass surface, comprising: a)immobilizing silicon rubber tubing on a glass surface by using of silicon rubber cement, b)overlapping the glass prepared in a) on a glass to be used in synthesizing oligonucleotides, c) injecting coupling solutions through the channel formed by overlapping to synthesize oligonucleotides in the specific site, and d)rotating sequentially silicon rubber tubing to synthesize oligonucleotides in the rest sites which is blocked(Southern, E. M., Maskos, U. and Elder, J. K. (1992) Genomics 13, 1008-1017; Maskos. U. and Southern, E. M. (1992) Nucleic Acids Res. 20, 1675-1678; Maskos, U. and Southern, E. M. (1993) Nucleic Acids Res. 21, 2267-2268; Williams, J. C., Case-Green, S. C., Mir, K. U. and Southern, E. M. (1994) Nucleic Acids Res. 22, 1365-1367).
Generally, oligonuclotides can be synthesized on a glass surface directly by using photosensitive oligonucleotide synthesis which is applied to DNA sequencing. In the above method, a free hydroxyl group is formed onto the surface which induced photosensitive protector from 5′ hydroxyl group by light emitted through shield mask, and then deoxynucleosides which are protected after forming free hydroxyl group are linked on the surface(Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M. T., Holmes, C. P. and Foder, S. P. A. (1994) Proc. Natl. Acad. Sci. USA 91, 5022-5026; Sapolsky, R. J. and Lipshutz, R. J. (1996) Genomics 33, 445-456; Hoheisel, J. D. (1997) Trends in Biotechnology 15, 465-469; Afftmetrix corp.).
The process for immobilizing oligonucleotides on polyacrylamide of which amide moiety is replaced with hydrazide group, has been developed by Russian scientists. In the above process, 3′-methyluridine at 3′-end of oligonucleotides is activated by sodium periodite(NaIO
4
) to form dialdehyde groups. Microchip for arranging oligonucleotides in the gel(100×100×20 &mgr;m) through the above method has been disclosed(Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4913-4918; Parinov, S., Barsky, V., Yershov, G., Kirillov, E., Timofeev, E., Belgovsky, A. and Mirzabekov, A. (1996) Nucleic Acids Res. 24, 2998-3004). Also, the method for immobilizing the oligonucleotides on the polypropylene film(PP-NH
2
), which are aminized by phosphoramidite-based synthesizing process has been disclosed(Matson, R. S., Rampal, J., Pentoney, S. L., Jr., Anderson, P. D. and Coassin, P. (1995) Analytical Biochemistry 224, 110-116). Another method for arranging oligonucleotides is to immobilize 3′-amino altered oligonucleotides on a thin film of silicone dioxide(SiO
2
), which is formed on a surface of the silicone chip, during the nucleic acid hybridization. In this method, the 3′-amino altered oligonucleotide is fixed onto the thin film of silicone dioxide(SiO
2
) through an epoxy ring-opening reaction of 3′-amino linkage with a epoxysilane monolayer, which is prepared by treating with 3′-glycidoxypropyltrimethoxysilane(Lamture, J. B., Beattie, K. L., Burke, B. E., Eiggers, M. C., Ehrlich, D. J., Fowler, R., Hollis, M. A. Kosicki, B. B., Reich, R. K., Smith, S. R., Varma, R. S. and Hogan, M. E. (1994) Nucleic Acids Res. 22, 2121-2125). Moreover, a method for inducing covalent bonds has been disclosed. In this method, the covalent bonds are induced by spotting oligonucleotides tailed with homopolymers(dTTP), to activate the thymine base of the oligonucleotide by Ultraniolet(UV) irradiation(Saiki, R. K., Walsh, P. S., Levenson, C. H. and Erlich, H. A. (1989) Proc. Natl. Acad. Sci. USA 86, 6230-6234). Further, a method improved from the above method has been disclosed. This method enables to make a more stable bond by forming amide bone between the amino-linker combined oligonucleotide and the carboxyl groups of the nylon membranes, and to increase efficiency of hybridization (Zhang, Y., Coyne, M. Y., Will, S. G., Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3922). The hybridization techniques by adding a radioactive labeled or non-radioactive labeled target DNA to the DNA chip which is developed by methods described above, are successfully used to detect RAS point mutation, cystic fibrosis deletion, and other various point mutation detection as well as DNA sequencing (Cantor, C. R., Mirzabekov, A. and Southern, E. (1992) Genomics 13, 1378-1383; Zhang Y., Coyne, M. Y., Will, S. G. Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3933; Hacia, J. J., Brody, L. C., Chee, M. S., Fodor, S. P. A. and Collins, F. S. (1996) Nature Genetics 14, 441-447; Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittman, M. and Davis, R. W. (1996) Nature Genetics 14, 450-456; Sosnowski, R. G., Tu, E., Butler, W. F., O'Connel, J. P. and Heller, M. J. (1997) Proc. Natl. Acad. Sci. USA 94, 1119-1123). These methods can be very effective, especially in genetic mutation tests and genetic polymorphism research (Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shai, N., Shen, N., Yang, R. and Fodor, S. P. A. (1995) Biotechniques 19, 442-447; Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. (1995) Science 270, 467-470; Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C. Stern, D., Winkler, J., Lock, D. J., Morris, M. S. and Fodor, S. P. A. (1996) Science 274, 610-614; DeRisi, J., Penland, L. and Brown, P. O.; Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A. and Trent, J. M. (1996) Nature Genetics 14, 457-460). Also, it is predicted that the above methods can be used as ideal tools for diagnosis of infectious and genetic/hereditary diseases as well as mutant analysis such as neoplasm, HLA typing and the like in view of their sensitivity, unity and reproducibility. (Saiki. R. K., Walsh, P. S., Levenson, C. H. and Erlich, H. A. (1989) Proc. Natl. Acad. Sci. USA 86, 6230-6234; Zhang, Y., Coyne, M. Y., Will, S. G., Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3933).
Methods for an oligonucleotide arrangement on a glass surface through direct photolithographic synthesis or other synthetic methods are very difficult requiring high-level techniques. One of the problems is that products on solid surface is relatively low, and thus requires further future consideration for adequate and appropriate quantitativ

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