Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-20
2003-12-02
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06656682
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid-immobilized substrate. More specifically, the present invention relates to a nucleic acid-immobilized substrate in which nucleic acids are securely immobilized on a carrier in the form of fine dots, useful as a DNA array and so forth.
Currently, the following two methods are mainly used for the preparation of a nucleic acid-immobilized substrate in which wherein nucleic acids are immobilized as fine dots on a carrier, for use as a DNA chip, DNA array or the like:
(1) a method utilizing immobilization of nucleic acids by physical adsorption on a base material coated with poly-L-lysine, which is used as a carrier (WO95/35505, International Patent Publication in Japanese (Kohyo) No. 10-503841/1998), and
(2) a method comprising DNA synthesis on a base material (WO97/10365).
However, a nucleic acid-immobilized substrate manufactured by the above method (1) has a drawback that, when it is used for hybridization, nucleic acids may be dropped off from the substrate, in particular, during operation processes, which leads to reduction of detection sensitivity, fluctuation of results, i.e., a problem of reproducibility and so forth. Further, as for the efficiency in immobilization of nucleic acids by the method (1), it has a drawback that short nucleic acids of about 300-mer or less such as oligomers cannot be immobilized efficiently, although longer nucleic acids can be immobilized without any particular problems.
Further, the above method (2) requires special apparatuses and reagents for synthesizing DNA on a base material and cannot be readily employed by everyone. Further, nucleic acids to be synthesized are limited to those of about 25-mer or less. Furthermore, nucleic acids longer than 10-mer cannot so easily be synthesized.
Thus, the conventional methods have problems not only of having difficulty in preparing a nucleic acid-immobilized substrate to immobilize nucleic acids of from 10-mer to 300-mer, but also of being unable to securely immobilize nucleic acids of other lengths or being unable to immobilize nucleic acids by using a simple apparatus.
SUMMARY OF THE INVENTION
The present invention has been accomplished from the above viewpoints, and an object thereof is to provide a nucleic acid-immobilized substrate in which nucleic acids are securely immobilized on a carrier in the form of fine dots irrespective of their length, and which can be prepared by using a simple apparatus.
As a result of efforts dedicated by the present inventors to achieve the above object, it was found that, if nucleic acids were immobilized through a carbodiimide group on a carrier composed of a base material carrying a compound having the carbodiimide group, nucleic acids could be securely immobilized on the carrier in the form of fine dots irrespective of their length. Also, it was found that, if nucleic acids were immobilized through an isocyanate group on a carrier composed of a base material carrying a compound having the isocyanate group, nucleic acids could be securely immobilized on the carrier in the form of fine dots irrespective of their length. Thus, the present invention has been accomplished.
The followings are provided by the present invention.
(1) A nucleic acid-immobilized substrate which comprises a carrier comprising a base material and a compound having a carbodiimide group carried by the base material, and the same kind or different kinds of nucleic acids immobilized in the form of dots through the carbodiimide group at a plurality of sites on the carrier (also referred to as “carbodiimide carrier” hereafter).
(2) The nucleic acid-immobilized substrate according to (1), wherein the dots each have a substantially circular shape and a diameter of from 10 to 3000 &mgr;m.
(3) The nucleic acid-immobilized substrate according to (1), wherein the nucleic acids have a chain length of from 10 to 300 nucleotides.
(4) The nucleic acid-immobilized substrate according to (1), wherein the compound having the carbodiimide group is carried on a surface of the base material through a covalent bond.
(5) The nucleic acid-immobilized substrate according to (1), wherein number of the dots in which nucleic acids are immobilized is 10 to 10,000 per cm
2
of the substrate.
(6) A nucleic acid-immobilized substrate which comprises a carrier comprising a base material and a compound having an isocyanate group carried by the base material, and the same kind or different kinds of nucleic acids immobilized in the form of dots through the isocyanate group at a plurality of sites on the carrier (also referred to as “isocyanate carrier” hereafter).
(7) The nucleic acid-immobilized substrate according to (6), wherein the dots each have a substantially circular shape and a diameter of from 10 to 3000 &mgr;m.
(8) The nucleic acid-immobilized substrate according to (6), wherein the nucleic acids have a chain length of from 10 to 300 nucleotides.
(9) The nucleic acid-immobilized substrate according to (6), wherein the compound having the isocyanate group is carried on a surface of the base material through a covalent bond.
(10) The nucleic acid-immobilized substrate according to (6), wherein number of the dots in which nucleic acids are immobilized is 10 to 10,000 per cm
2
of the substrate.
According to the present invention, there is provided a nucleic acid-immobilized substrate in which DNAs are stably immobilized. Since nucleic acids can be immobilized on the substrate of the present invention without any limitation concerning the number of chains or the length of nucleic acids, various kinds of nucleic acids can simultaneously be handled on the same base material.
Furthermore, since nucleic acids are securely bound to the carrier through covalent bonds, the nucleic acid-immobilized substrate can be useful for use as a DNA chip of excellent reproducibility and quantification characteristics.
DETAILED DESCRIPTION OF THE INVENTION
(1) Carrier
The carrier used for the nucleic acid-immobilized substrate of the present invention is provided for immobilizing nucleic acids and comprises a base material and a compound having a carbodiimide group or an isocyanate group (also referred to simply as “carbodiimide compound” or “isocyanate compound” hereafter, respectively) carried by the base material.
A. Carbodiimide Carrier
(1) Base Material
The base material used for the present invention serve's as a support for the aforementioned carrier and is not particularly limited so long as it is basically insoluble in a solvent and is in a solid or gel state at an ordinary temperature or within a temperature range around the ordinary temperature (0 to 100° C.). The expression that the base material is insoluble in a solvent means that the base material is substantially insoluble in various solvents such as aqueous solvents and organic solvents used in various processes when the carbodiimide compounds are provided on the base material and nucleic acids are immobilized on the base material as a carrier, as will be described later, and then it is used as, for example, a DNA chip.
Materials used for such a base material of the carrier include, specifically, plastics, inorganic polymers, metals, natural polymers, ceramics and the like.
Examples of the plastics include, specifically, polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenol resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyethylene fluoride, polyimide, acrylic resin and so forth. Examples of the inorganic polymers include glass, quartz, carbon, silica gel, graphite and so forth. Examples of the metals include metals that are solid at an ordinary temperature such as gold, platinum, silver, copper, iron, aluminum, magnet and paramagnet. Examples of the natural polymers include cellulose, cellulose derivatives, chitin, chitosan, alginic acid, alginic acid salts and so forth. Examples of the ceramics include apatite, alumina, silica, silicon carbide, silicon nitride, boron carbide and so forth.
The base material can be in the form of,
Ichihara Tatsuo
Matsumura Yoshiyuki
Shiohata Namiko
Suzuki Osamu
Kilpatrick & Stockton LLP
Nisshinbo Industries Inc.
Siew Jeffrey
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