Method of immobilizing water-soluble bioorganic compounds on a c

Details Method of immobilizing water-soluble bioorganic compounds on a c Method of immobilizing water-soluble bioorganic compounds on a c

1995-06-02

1998-04-21

Marschel, Ardin H.

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

435 6, 4352872, 4352879, 530300, 536 221, 536 231, 536 253, 536 254, C12M 100, C12Q 168, A61K 3800, C07H 1900

Patent

active

057417008

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates in general to molecular biology and more specifically to a method for immobilizing water-soluble bioorganic compounds onto a capillary-porous carrier.


BACKGROUND OF THE INVENTION

Known in the present state of the art are quite a number of chemical methods for immobilizing water-soluble bioorganic compounds, e.g., proteins, peptides, and DNA fragments (oligonucleotides and polynucleotides) on capillary-porous carriers. The methods are based on establishing covalent bonds between the bioorganic compound and the carrier. Used as the carrier are: cellulose, carboxymethylcellulose, agarose, dextran, polyaminopolystyrene, polyacrylamides and their derivatives, and others.
One state-of-the-art method for immobilizing oligonucleotides on a capillary porous carrier, namely on a gel matrix (SU, A No. 1,794,088), consists in that the drops of a solution of oligonucleotides are applied to an air-dried gel matrix with the aid of a micromanipulator provided with a dispenser, whereupon the matrix with the solution of oligonucleotides applied thereto is placed in a wet chamber for 4 hours till completion of the reaction of bonding oligonucleotides to gel. Then the matrix is dried for 0.5 hour in the open air, washed with a hybridization buffer (1M NaCl, 10 mM Na.sub.3 PO.sub.4.7H.sub.2 O, pH 7.0, 1 mM ethylenediaminetetraacetic acid), rinsed with water, and stored dry at minus 20.degree. C. Oligonucleotides are immobilized on a gel matrix, said gel being applied to the substrate, as areas (square cells) spaced from one another.
The chemical reaction of oligonucleotide-to-gel bonding is conducted as follows. Used as a linking agent is 3-methyluridine bound by a 5'-3' internucleotide phosphodiester bond to the oligonucleotide to be immobilized. 3-methyluridine is given preference due to its inability of forming strong hydrogen bonds with any naturally occurring bases.
Prior to applying the gel to the matrix, oligonucleotides containing 3-methyluridine at their 3'-end are oxidized with 1 mM sodium periodate for one hour at room temperature, precipitated with 10 volumes of 2% LiClO.sub.4 (lithium perchlorate) in acetone, and dissolved in water.
Oxidation of oligodeoxynucleotide results in formation of a derivative carrying a dialdehyde group at the 3'-end. Before use, the gel matrix is treated with 50% hydrazine whereby a part of the amide groups are substituted by the hydrazide ones which readily react with 3'-dialdehyde to yield a stable morpholine derivative.
The course of immobilization is monitored against the marker (5'-.sup.32 P) introduced with the aid of kinase, into the oligonucleotides being immobilized. The immobilization yield (i.e., the percentage of the oligonucleotide irreversibly bonded with the gel) is close to 80%.
The aforediscussed method, however, has a restriction on its application whenever it is necessary to immobilize a great number (above 10) of various nucleotides, contained in microvolumes (up to tens of nanoliters) of solutions located in the cells (measuring up to 100 .mu.m) of a dense polyacrylamide gel micromatrix, with the cell spacing up to 200 .mu.m when strictly single-type nucleotides are to be placed in each cell. In this case standard conditions for the reaction of covalent bonding of oligonucleotides with the carrier are difficult to attain in all the cells of the matrix, due to inescapable partial evaporation of solutions in some of the cells long before the reaction is completed and often already in the process of applying the solutions and during transfer of the micromatrix to a wet chamber. Thus, the quality of immobilization and, accordingly, that of the micromatrix are affected, the consumption of expensive reagents is increased and the process becomes more costly.
Furthermore, the moisture-exchange process on the matrix surface becomes difficult to control after the matrix has been placed in the wet chamber where water condensate may fall abundantly from the vapor-gaseous phase upon the micromatrix surface and connect the

REFERENCES:
patent: 5216126 (1993-06-01), Cox et al.
patent: 5279821 (1994-01-01), Hirayama et al.
Matthews et al. "Review Analytical strategies for the use of DNA probes" Analytical biochemistry, vol. 169, pp. 1-25, Feb. 1988.

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