Nucleic acid-immobilized substrate

Details Nucleic acid-immobilized substrate Nucleic acid-immobilized substrate

2000-11-29

2002-12-24

Riley, Jezia (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S025320, C536S026600

Reexamination Certificate

active

06498245

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a nucleic acid-immobilized substrate. More particularly, it relates to a nucleic acid-immobilized substrate that can efficiently immobilize nucleic acids on a carrier in a simple manner as well as a production method therefor and an analysis method by utilizing it.
2. Description of the Related Art
As the method for producing nucleic acid-immobilized substrates comprising a carrier on which nucleic acids and so forth are immobilized, which are used for DNA tips and so forth, the following two kinds of methods are mainly used:
(1) a method of immobilizing nucleic acids by physical adsorption using a base material coated with poly-L-lysine etc. as a carrier (International Patent Publication in Japanese (Kohyo) No. 10-503841/1998), and
(2) a method of synthesizing DNA on a base material (WO97/10365).
Among the aforementioned methods, however, the method described in International Patent Publication in Japanese No. 10-503841/1998 has a drawback that if hybridization is performed by using this method, nucleic acids are dropped off from the substrate especially during operational steps, and consequently detection sensitivity may be reduced and results may be varied to cause bad reproducibility. Furthermore, this method also suffers from a drawback that a short chain nucleic acid of about 50-mer or less such as oligomers cannot be efficiently immobilized, although a long chain nucleic acid can be immobilized by this method.
Further, the method described in WO97/10365 requires an extremely special apparatus and extremely special regents, because DNA is synthesized on a base material. Thus, it is not a method that can be easily performed by anyone. Moreover, it also suffers from a drawback that nucleic acids that can be synthesized are limited to those of about 25-mer or shorter.
SUMMARY OF THE INVENTION
The present invention was accomplished in view of the aforementioned problems, and its object is to provide a nucleic acid-immobilized substrate on which nucleic acids are firmly immobilized in fine dot areas without reference to chain length of the nucleic acids, which enables efficiently introducing the nucleic acids onto a base material in a simple manner and can be produced with a simple apparatus.
The inventors of the present invention found that if nucleic acids are immobilized on a carrier that comprises a base material carrying compound having one or more alkylating groups through the intermediary of the alkylating group, the nucleic acids can firmly be immobilized on the carrier in fine dot areas without reference to chain length of the nucleic acids, and the aforementioned object can be achieved. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A nucleic acid-immobilized substrate having a carrier and identical or different nucleic acids,
wherein the carrier comprises a base material and compound on the base material, the compound having one or more alkylating groups, and the nucleic acids are immobilized in a plurality of dot-like areas on the carrier through the intermediary of the alkylating group.
(2) The nucleic acid-immobilized substrate according to (1), wherein the compound having the alkylating groups consists of nitrogen yperite.
(3) The nucleic acid-immobilized substrate according to (1) or (2), wherein the carrier has a structure represented by the following general formula (I):
M—R
n
—G  (I)
[In the formula, M represents the compound having one or more alkylating groups;
R represents a functional group selected from the group consisting of —NH—, —CH
2
—, —NHCO—, —CONH—, —O—, —S—, —N(R
1
)— (R
1
represents a normal, cyclic, or branched saturated aliphatic hydrocarbon group having 1-20 carbon atoms or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms),
(R
2
and R
3
independently represent a hydrogen atom, a normal or branched saturated aliphatic hydrocarbon group having 1-20 carbon atoms or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, a cycloalkyl group, an aryl group or an aralkyl group, which may have one or more substituents; provided that one of R
2
and R
3
represents a hydrogen atom, the other represents a normal or branched saturated aliphatic hydrocarbon group having 1-20 carbon atoms or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, a cycloalkyl group, an aryl group or an aralkyl group, which may have one or more substituents; R
2
and R
3
may be also bonded together to form a nitrogen-containing heterocyclic group that may contain an oxygen atom), —COO—, —OCO—, —NHSO
2
—, —NHC(S)NH— and —SO
2
NH—;
n represents an integer of 0-20;
when two or more groups of R exist, they may be identical to or different from each other;
G represents the base material or a polymer that can be adhered to the base material].
(4) A carrier for immobilizing nucleic acids, which comprises a base material and compound carried on the base material, the compound having one or more alkylating groups.
(5) A method for producing a carrier for immobilizing nucleic acids, the carrier comprising a base material and compound carried on the base material, the compound having one or more alkylating groups,
which comprises the step of getting the compound having two or more alkylating groups or the compound having one or more alkylating groups and one or more functional groups that are not alkylating groups, to covalently bond with functional groups on a surface of the base material having the functional groups can be covalently bonded with the alkylating groups or with the functional groups that are not alkylating groups,
while leaving at least one of the alkylating groups of the compound.
(6) A method for producing a nucleic acid-immobilized substrate, which comprises the step of bringing nucleic acids into contact with a carrier for immobilizing nucleic acids,
wherein the carrier comprises a base material and compound carried on the base material, the compound having one or more alkylating groups.
(7) A method for detecting a nucleic acid by hybridization using a nucleic acid labeled with a labeling substance,
wherein the nucleic acid-immobilized substrate according to any one of (1) to (3) is used.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereafter, embodiments of the present invention will be explained in detail.
<1> Carrier
The carrier used for the nucleic acid-immobilized substrate of the present invention is for immobilizing nucleic acids, and comprises a base material and compound carried on the base material, the compound having one or more alkylating groups (this may also be referred to as “alkylating reagent” hereafter).
(1) Base material
The base material used for the present invention serves as a support of the aforementioned carrier, and it is not particularly limited so long as it basically composed of a material that is insoluble in a solvent and is in a state of solid or gel at an ordinary temperature or within a temperature range around it (0-100° C.). The definition that the base material is insoluble in a solvent means that the base material is substantially insoluble in various aqueous and organic solvents used for process steps where the alkylating reagent is provided on the base material to be carried on the base material as described hereinafter, then nucleic acids are immobilized thereon as the carrier, and then the substrate is used as a DNA tip or the like.
Specific examples of the material of the carrier base material include plastics, inorganic polymers, metals, natural polymers, ceramics and so forth.
Specific examples of the plastics include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenol resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, fluorinated polyethylene, polyimide, acrylic resin and so forth. Specific examples of the inorganic polymers include glass, crystal, carbon, silica gel, graphite and so forth. Specific examples of the metals include gold, pl

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