Gene detection method

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GENE DETECTION METHOD

This application is a Continuation-In-Part of U.S. application Ser. No. 07/766.064. filed on Sep. 27.1991; claiming foreign priority to Japanese Application 2-259011, filed on 5 Sep. 28. 1990. claiming foreign priority to Japanese Application 3-90879. filed on Apr. 22. 1991, claiming foreign priority to Japanese Application 3-191868, filed on Jul. 31. 1991, claiming foreign priority to Japanese Application 91308770.6. filed on Sep. 26. 1991. claiming foreign prior- to ity to Japanese Application 4-242397, filed on Sep. 10,1992.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel gene detection method to detect a certain gene specifically and a device for such detection.

2. Description of the Related Art

A genetic information stored in DNA is expressed as a 20 protein or an enzyme through mRNA. By the effects of such protein or enzyme, various compounds necessary to maintain the vital actions are biosynthesized and metabolized. Thus a life is present as a dynamic equilibrium system of various substances controlled by genes. 25

There are 50 to 100 thousands of human genes. When some of them involve abnormality or change, such as defect or duplication, the characteristics, types and amounts of the proteins synthesized are changed, resulting in the poorly balanced biosystem, which may cause diseases. Thus, by 30 detecting known pathogenic genes, the diseases may be identified or prevented. Such diagnosis based on the genes themselves has been developed as a results of the recently advancing technology of gene engineering, and is called as gene diagnosis. 35

When compared with conventional diagnostic methods, the gene diagnosis has characteristics as mentioned below.

Considering the mechanisms of gene expression, it can be presumed that changes in genes occur prior to almost all biochemical changes. Therefore, gene diagnosis by means of 40 detecting the genetic change enables the diagnosis or prognosis prior to development of a disease which is one of phenotypes. Accordingly, the dignosis and prognosis can be conducted before (he development, in the latent period or at the earliest stage of the disease. This is the primary charac- 45 teristic. As the secondary characteristic, gene diagnosis relating to the genetic diseases is independent from the organs or tissues to be analyzed since all genes in a living body are the same. This is particularly important in the diagnosis in fetus. Thus, this secondary characteristic 50 enables the diagnosis simply by sampling amniotic fluid from a pregnant woman and analyzing the fetal cells suspending in the amniotic fluid.

Procedure of gene diagnosis conventionally employed is J5 summarized as follows.

Genes are extracted from a samples and cleaved, if necessary, by appropriate restriction enzymes, and then subjected to electrophoresis and southern blotting.

Then a nucleic acid probe (usually radiolabelled) having go the base sequence complementary to the gene to be detected is hybridized to the blotted gene. Subsequently, the hybridized nucleic acid probe is detected by exposing an X-ray film to the radiation emitted from the labeled probe at lower temperature to confirm the presence of the gene. 65

The conventional detection method mentioned above involves the limitation of the place of diagnosis due to the

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use of radioisotopes and should be conducted with sufficient care of handling reagents. For the purpose of reducing such inconvenience, safe labeling agents substituting the radioisotopes are being developed and several detection methods, in which probes are utilized, such as avidin-biotin bond method or enzymatic or fluorescent method and the like have already been suggested. However, these methods can not achieve the sensitivity superior to that of the method using radioisotopes. They also involve the problems of the time period required to detecting the gene as long as 2 or 3 days as well as complicated procedure of determination, on the other hand, quantification of a certain antigen or antibody present in a sample generally employs radioimmunoassay (RIA). However, RIA requires special instruments and authorized operators therefor capable of handling radioisotopes since this method also employs radioisotopes similarly as in the gene diagnosis methods mentioned above. In addition, waste disposal in this method should be done with particular care. As one of the other analytic methods, Immunoelectrophoresis, which requires a long period for determination and has a poor sensitivity, can be suggested, although this method is not applicable in case of the samples containing only trace amount of test substance.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a method which is excellent in safety and convenience and is capable of detecting a certain gene at a high sensitivity in a reduced time period.

Accordingly, the gene detection method of the present invention is the method wherein a single stranded nucleic acid probe having a base sequence complementary to the gene to be detected is reacted with a gene sample denatured into a single stranded form and then the nucleic acid probe hybridized with the gene is detected to confirm the presence of the gene, characterized in that:

the nucleic acid probe is immobilized onto a carrier sensitive to a physical change;

a double stranded nucleic acid recognizing substance capable of binding specifically to a double stranded nucleic acid and being active physicochemically is added to the reaction system of the nucleic acid probe and the gene sample; and, the double stranded nucleic acid recognizing substance bound to the double stranded nucleic acid formed by conjugation of the nucleic acid probe and the gene to be detected is detected by means of physicochemical determination using the carrier, whereby detecting the presence of the nucleic acid probe hybridized with the gene to be detected.

Another object of the present invention is to provide a gene detection device which is excellent in safety and convenience and is capable of detecting a certain gene at a high sensitivity in a reduced time period.

Accordingly, the gene detection device of the present invention is the device comprising:

a gene detection sensor having a nucleic acid probe immobilized onto the surface of a carrier sensitive to a physical change; a transportation means to transport the gene detection sensor;

a reaction bath to store a sample solution containing the gene sample denatured into a single stranded form, a double stranded nucleic acid being formed in said reaction bath on the gene sensor by hybridization of the gene sample and the nucleic acid probe immobilized on the surface of the gene sensor;

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a temperature control means to control the temperature of

the sample solution; a washing means to remove unreacted gene sample by washing the gene sensor after hybridization of the nucleic acid probe with the gene sample; and a detection bath to store a double stranded nucleic acid recognizing substance, the double stranded nucleic acid recognizing substance being reacted with the double stranded nucleic acid formed on the surface of the gene sensor in said detection bath, whereby binding the double stranded nucleic acid recognizing substance with the double stranded nucleic acid in order to detect a physical change generated by the bound double stranded nucleic acid recognizing substance. Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate a presently preferred embodiment of the invention, and together with the general description given above and the detailed description of the preferred embodiment given below, serve to explain the principles of the invention.

FIG. 1 shows a diagram schematically representing an example of the automatic gene detection device according to the present invention.

FIG. 2 shows a perspective view of another embodiment of the reaction bath and a gene sample purification device of the present automatic gene detection device shown in FIG. 1.

FIG. 3 shows a perspective view of an example of the temperature controller in the present automatic gene detection device shown in FIG. 1.

FIG. 4 shows a diagram schematically representing an example of the present automatic gene detection device utilizing electrochemiluminescene.

FIG. 5 shows a plan view of the temperature gradient reaction vessel used for screening a nucleic acid probe in Example 29.

FIG. 6 shows a perspective view of a basic plate for immobilizing a nucleic acid probe used in Example 30.

FIG. 7 shows a diagram schematically representing an example of the autonomic gene detection device utilizing carrier with a sample nucleic acid immobilized thereon.

FIG. 8 shows a diagram schematically representing an example of the autonomic gene detection device utilizing a carrier with nucleic acid sample immobilized thereon and electrochemiluminescence.

FIG. 9 shows a schematic view of the determination results in Example 30. in which a shaded box of a grid shows a positive result.

FIG. 10 shows a schematic view of the determination results in Example 31. in which a shaded box of a grid shows a positive result.

DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENTS

In the present invention, the term "double stranded nucleic acid recognizing substance" means a substance

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which recognizes and binds specifically to a double stranded nucleic acid. Such substances are, for example, intercalating agents and biopolymers capable of recognizing double stranded nucleic acid.

5 The intercalating agents are characterized by their tendency to intercalate specifically to double stranded nucleic acid such as double stranded DNA. These intercalating agents have in their molecules a flat intercalating group such as phenyl group, which intercalates between the base pairs

10 of the double stranded nucleic acid, whereby binding to the double stranded nucleic acid. Most of the intercalating agents are optically active and some of them are used in qualification of nucleic acids. Certain intercalating agents exhibit electrode response. Therefore, determination of

15 physical change, especially optical or electrochemical change, may serve to detect the intercalating agents bound to a double stranded nucleic acid.

Electrochemically or optically active intercalating agents useful in the present invention are. but are not limited to,

20 ethidium. ethidium bromide, acridine, aminoacridine. acridine orange, proflavin. ellipticine, actinomycin D, daunomycin, mitomycin C, HOECHST 33342. HOECHST 33258, aclarubicin. DAPI, Adriamycin, pirarubicin. actinomycin, tris (phenanthroline) zinc salt, tris

25 (phenanthroline) ruthenium salt, tris (phenantroline) cobalt salt, di (phenanthroline) zinc salt, di (phenanthroline) ruthenium salt, di (phenanthroline) cobalt salt, bipyridine platinum salt, terpyridine platinum salt, phenanthroline platinum salt, tris (bipyridyl) zinc salt, tris (bipyridyl) ruthenium salt,

30 tris (bipyridyl) cobalt salt, di (bipyridyl) zinc salt, di (bipyridyl) ruthenium salt, di (bipyridyl) cobalt salt, and the like. Other intercalating agents which are useful are those listed in Published Japanese Patent Application No. 62-282599.

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In addition to the intercalating agents which are reversibly reacted themselves during oxidation-reduction reaction as listed above, the determination of electrochemical change using an electrode may employ a metal complex containing

^ as a center metal a substance capable of undergoing electrically reversible oxidation-reduction reaction, namely, a metallo intercalater. Such metallo intercalaters include for example tris (phenanthroline) zinc salt, his (phenanthroline) ruthenium salt, tris (phenanthroline) cobalt salt, di

45 (phenthroline) zinc salt, di (phenanthroline) ruthenium salt, di (phenanthroline) cobalt salt, bipyridine cobalt salt, terpyridine platinum salt, phenanthroline platinum salt, tris (bipyridyl) zinc salt, tris (bipyridyl) ruthenium salt, tris (bipyridyl) cobalt salt, di (bipyridyl) zinc salt, di (bipyridyl)

50 ruthenium salt, di (bipyridyl) cobalt salt and the like. Although the intercalating agents are not limited to those listed above, the complexes which or whose center metals have oxidation-reduction potentials not lower than or covered by that of nucleic acids are less preferable.

55 By using the intercalating agents capable of undergoing electrochemically reversible oxidation-reduction reaction, it is possible to determine the oxidation-reduction current repetitively. Accordingly, it is possible to conduct potential scanning several to several hundreds times and to sum up the

go values of the signals obtained, whereby enabling the amplification of the signals, resulting in a higher sensitivity of the detection.

When conducting the detection of the gene using an electrode, an intercalating agent exhibiting electrochemilu65 minescence may also be employed. Such intercalating agents are, but are not limited to, for example, luminol. lucigenin. pyrene, diphenylanthracene rubrene and acri