JP4034818B1 - Carrier for gene detection and its use to detect the effectiveness of interferon therapy - Google Patents

Abstract

The present invention provides a means for predicting whether or not interferon therapy is effective for a patient before treatment.
A carrier for gene detection, a method for detecting the effectiveness of interferon therapy for an individual, a device for gene detection, and a kit for detecting the effectiveness of interferon therapy are provided.
[Selection] Figure 1

Description

  The present invention relates to a carrier for gene detection, a method for detecting the effectiveness of interferon therapy for an individual, a device for gene detection for detecting the effectiveness of interferon therapy for an individual, and gene detection for detecting the effectiveness of interferon therapy Relates to the kit.

  In recent years, the number of people infected with hepatitis C virus (hereinafter referred to as HCV) has increased rapidly, which has become a major social problem.

  As with other viral diseases, it has been shown that interferon therapy has certain effects on hepatitis C, but interferon therapy is not effective for all infected individuals. There are quite a few infected people for whom the therapy does not work. Continuing interferon therapy for infected patients who have no effect on interferon therapy not only causes side effects such as fever and anemia in the infected person, but also delays the start of other treatments.

  For this reason, it has been desired to predict whether interferon therapy is effective for an HCV-infected person to be treated before treatment, but until now, it is impossible to make such a prediction at all. there were.

  In view of the above circumstances, an object of the present invention is to provide a means for predicting whether or not interferon therapy is effective for a patient before treatment.

  The above-mentioned subject is achieved by the following present invention.

According to the first aspect of the present invention,
A substrate;
Comprising a polynucleotide immobilized on the substrate,
The polynucleotide is
(at) a polynucleotide represented by SEQ ID NO: 1 in the sequence listing below,
(bt) a modified polynucleotide in which one or several nucleotides except for position 455 in the polynucleotide shown in (at) are deleted, substituted, or added;
(ct) a polynucleotide comprising the sequence shown in positions 441 to 455 of SEQ ID NO: 1,
(dt) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 1,
(et) A gene detection carrier comprising a polynucleotide selected from the group consisting of their complementary strands is provided.

According to a second aspect of the present invention,
A substrate;
Comprising a polynucleotide immobilized on the substrate,
The polynucleotide is
(ag) a polynucleotide represented by SEQ ID NO: 2 in the sequence listing below,
(bg) a modified polynucleotide in which one or several nucleotides other than position 455 in the polynucleotide shown in (ag) are deleted, substituted, or added;
(cg) a polynucleotide comprising the sequence shown in positions 441 to 455 of SEQ ID NO: 2,
(dg) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 2,
(eg) Provided is a gene detection carrier comprising a polynucleotide selected from the group consisting of their complementary strands.

According to the third aspect of the present invention,
A substrate;
Comprising a polynucleotide immobilized on the substrate,
The polynucleotide is
(aa) a polynucleotide represented by SEQ ID NO: 3 in the sequence listing below,
(ba) a modified polynucleotide in which one or several nucleotides except for position 455 in the polynucleotide shown in (aa) are deleted, substituted, or added;
(ca) a polynucleotide comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 3,
(da) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 3,
(ea) A gene detection carrier comprising a polynucleotide selected from the group consisting of their complementary strands is provided.

According to a fourth aspect of the present invention,
A substrate;
Comprising a polynucleotide immobilized on the substrate,
The polynucleotide is
(ac) a polynucleotide represented by SEQ ID NO: 4 in the sequence listing below,
(bc) a modified polynucleotide in which one or several nucleotides other than position 455 in the polynucleotide shown in (ac) are deleted, substituted, or added;
(cc) a polynucleotide comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 4,
(dc) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 4,
(ec) Provided is a gene detection carrier comprising a polynucleotide selected from the group consisting of their complementary strands.

  According to the fifth aspect of the present invention, there is provided a DNA chip using the gene detection carrier.

According to a sixth aspect of the present invention, there is provided a method for detecting the effectiveness of interferon therapy for an individual,
1) contacting a sample polynucleotide collected from the individual with the carrier for gene detection;
And 2) detecting a hybridization reaction between the sample polynucleotide and the polynucleotide immobilized on the gene detection carrier, and determining a nucleotide sequence of the polynucleotide in the sample.

According to the seventh aspect of the present invention, there is provided a method for detecting the effectiveness of interferon therapy for an individual,
1) contacting a probe polynucleotide with a carrier for gene detection in which a sample polynucleotide collected from the individual is immobilized on a substrate;
And 2) detecting a hybridization reaction between the sample polynucleotide immobilized on the substrate and the polynucleotide, and determining a base sequence of the sample polynucleotide.

The probe polynucleotide is
(at) a polynucleotide represented by SEQ ID NO: 1 in the sequence listing below,
(bt) a modified polynucleotide in which one or several nucleotides except for position 455 in the polynucleotide shown in (at) are deleted, substituted, or added;
(ct) a polynucleotide comprising the sequence shown in positions 441 to 455 of SEQ ID NO: 1,
(dt) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 1,
(et) their complementary strand
(ag) a polynucleotide represented by SEQ ID NO: 2 in the sequence listing below,
(bg) a modified polynucleotide in which one or several nucleotides other than position 455 in the polynucleotide shown above are deleted, substituted, or added;
(cg) a polynucleotide comprising the sequence shown in positions 441 to 455 of SEQ ID NO: 2,
(dg) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 2,
(eg) their complementary strand
(aa) a polynucleotide represented by SEQ ID NO: 3 in the sequence listing below,
(ba) a modified polynucleotide in which one or several nucleotides except for position 455 in the polynucleotide shown in (aa) are deleted, substituted, or added;
(ca) a polynucleotide comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 3,
(da) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 3,
(ea) their complementary strand
(ac) a polynucleotide represented by SEQ ID NO: 4 in the sequence listing below,
(bc) a modified polynucleotide in which one or several nucleotides other than position 455 in the polynucleotide shown in (ac) are deleted, substituted, or added;
(cc) a polynucleotide comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 4,
(dc) a polynucleotide comprising the sequence shown in positions 449 to 459 of SEQ ID NO: 4,
(ec) includes a polynucleotide selected from the group consisting of their complementary strands.

According to the eighth aspect of the present invention,
The gene detection carrier;
A sample containing a polynucleotide immobilized on the substrate and a labeled polynucleotide are brought into contact with each other, and the polynucleotide immobilized on the substrate and the labeled polynucleotide are hybridized. A reaction part to be placed under zeation reaction conditions;
There is provided a gene detection device for detecting the effectiveness of interferon therapy, comprising at least a label detection device for detecting a label attached to the labeled polynucleotide.

According to the ninth aspect of the present invention,
The gene detection carrier;
With the counter electrode,
A voltage application unit for applying a voltage between the gene detection carrier and the counter electrode;
The polynucleotide immobilized on the carrier for gene detection and a sample containing the polynucleotide are brought into contact with each other, and the polynucleotide immobilized on the substrate and the polynucleotide in the sample are subjected to hybridization reaction conditions. A reaction part to put,
For detecting the effectiveness of interferon therapy comprising the carrier for gene detection and a measurement unit for measuring an electrical signal generated between the counter electrode when a voltage is applied by the voltage application means after the hybridization reaction. An apparatus for gene detection is provided.

  According to a tenth aspect of the present invention, there is provided a kit for measuring the effectiveness of interferon treatment, comprising the gene detection carrier and a buffer.

  According to an eleventh aspect of the present invention, there is provided a kit for detecting the effectiveness of interferon treatment for an individual comprising the carrier for gene detection, a double-stranded recognizer, and a buffer. Provided.

  The present invention provided a means to predict before treatment whether interferon therapy is effective for a patient.

  Hereinafter, the present invention will be described in detail.

  The polynucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 are polynucleotides that include the promoter region of the human MxA gene and are present by the present inventors at position 455 of these polynucleotides. It has been found for the first time that single nucleotide polymorphism (hereinafter referred to as SNP) is involved in responsiveness to the effects of interferon therapy.

  An interferon-stimulated response element (hereinafter referred to as ISRE) is present at positions 441 to 456 of each polynucleotide.

  The base sequence of ISRE at positions 441 to 456 of SEQ ID NO: 1 is “GGTTTCGTTTCTG CTC” (SEQ ID NO: 5), which corresponds to the 15th position of ISRE (corresponding to position 455 of SEQ ID NO: 1. Position 455 in SEQ ID NO: 1 According to the usual notation where the transcription start site is +1, it corresponds to position −88) is thymine.

  The nucleotide sequence of ISRE at positions 441 to 456 of SEQ ID NO: 2 is “GGTTTCGTTTCTGCGC” (SEQ ID NO: 6), which corresponds to the 15th position of ISRE (corresponding to position 455 of SEQ ID NO: 2. Position 455 in SEQ ID NO: 2 is transcription. According to the usual notation where the start site is +1, it corresponds to position -88) is guanine.

  The base sequence of ISRE at positions 441 to 456 of SEQ ID NO: 3 is “GGTTTCGTTTCTGCAC” (SEQ ID NO: 7), which corresponds to the 15th position of ISRE (corresponding to position 455 of SEQ ID NO: 3. Position 455 in SEQ ID NO: 3 is According to the usual notation where the transcription start site is +1, it corresponds to position -88) is adenine.

  The base sequence of ISRE at positions 441 to 456 of SEQ ID NO: 4 is “GGTTTCGTTTCTGCCC” (SEQ ID NO: 8), which corresponds to the 15th position of ISRE (corresponding to position 455 of SEQ ID NO: 4. Position 455 in SEQ ID NO: 4 is transcription. According to the usual notation where the start site is +1, it corresponds to position -88) is cytosine.

  Hereinafter, position 455 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 is referred to as a SNP site throughout this specification.

  These regions other than the SNP site of ISRE are common to each sequence. HCV-infected individuals with ISRE (SEQ ID NO: 5) whose 15th nucleotide of these ISREs is thymine are effective against interferon therapy, whereas ISRE (SEQ ID NO: 5) whose 15th nucleotide is thymine It has been epidemiologically demonstrated by the inventors that interferon therapy is not effective for those who do not have HCV.

  That is, as detailed in the Examples, a HCV-infected person having a polynucleotide of SEQ ID NO: 1 in which the nucleotide at position 455 is guanine is homozygous (hereinafter abbreviated as G / G homozygous) An HCV-infected person having a heterozygote (hereinafter abbreviated as G / T heterozygote), or a sequence having the polynucleotide of SEQ ID NO: 1 in which the nucleotide is thymine and the promoter region of SEQ ID NO: 1 in which nucleotide 455 is guanine It has been demonstrated that interferon therapy is not effective compared to HCV-infected persons having the polynucleotide of No. 1 in homozygosity (hereinafter abbreviated as T / T homo).

  Alternatively, an HCV-infected person (hereinafter abbreviated as non-T / non-T homo) having a promoter region of the MxA gene whose nucleotide at position 455 is not thymine is homozygous for T / non-T heterozygous or T / T homologous. Interferon therapy was shown to be ineffective compared to HCV-infected individuals. Non-T / non-T homo combinations include G / G, G / A, G / C, A / A, A / C, and C / C. T / non-T combinations include T / G, T / A, and T / C.

  Therefore, prior to the implementation of interferon therapy, for example, by determining the nucleotide of the SNP site in the ISRE of a polynucleotide containing the promoter region of the human MxA gene possessed by the HCV infected person, the interferon therapy is performed on the HCV infected person. The effectiveness of the can be detected.

  The gene detection carrier or the like of the present invention is a polynucleotide of SEQ ID NO: 1 comprising the SNP site and the base sequence of the SNP site being thymine as a probe for detecting the sequence of a nucleic acid chain of a polynucleotide extracted from a subject, Its fragments or their complementary strands are used.

  By using the carrier for gene detection of the present invention or the like, it can be examined before treatment whether or not the SNP site of the polynucleotide containing the promoter region of the human MxA gene possessed by the subject is thymine. Thereby, the effectiveness of interferon therapy for the subject can be predicted.

  In addition, the gene detection carrier of the present invention includes the SNP site as a probe for detecting the sequence of the nucleic acid chain of a polynucleotide extracted from a subject, and the polynucleotide of SEQ ID NO: 2 wherein the base sequence of the SNP site is g , A fragment thereof or a complementary strand thereof, or a polynucleotide of SEQ ID NO: 3 wherein the nucleotide sequence of the SNP site is a, a fragment thereof or a complementary strand thereof, or the nucleotide sequence of SEQ ID NO: 4 wherein the nucleotide sequence of the SNP site is c Polynucleotides, fragments thereof or their complementary strands are used.

  By using the carrier for gene detection of the present invention and the like, the sequence of the SNP site of the polynucleotide including the promoter region of the human MxA gene possessed by the subject can be examined, thereby confirming the effectiveness of interferon therapy for the subject. Can be predicted.

  In addition to hepatitis C, diseases to be treated with interferon therapy include glioblastoma, medulloblastoma, astrocytoma, cutaneous malignant melanoma, hepatitis B, renal cancer, multiple myeloma, hairy cell leukemia, chronic Myeloid leukemia, subacute sclerosing panencephalitis, viral encephalitis, systemic herpes zoster and chickenpox in immunosuppressed patients, nasopharyngeal undifferentiated cancer, viral inner ear infection with hearing loss, herpes keratitis, flattened condyloma, Conjunctivitis due to warts, adenovirus and herpes virus infection, genital herpes, cold sores, cervical cancer, cancerous pleural effusion, keratophyte cell carcinoma, basal cell carcinoma, δ type chronic active hepatitis and the like. Examples of interferons used in the interferon therapy include interferon α, β, and ω.

  The carrier for gene detection or the like of the present invention can be used to detect the effectiveness of interferon therapy before performing interferon therapy on a subject suffering from these diseases.

  Next, each aspect of the present invention will be described in more detail.

  First, the present invention provides a carrier for gene detection that can be used to detect the effectiveness of interferon therapy for patients suffering from diseases for which interferon therapy can be effective prior to the implementation of interferon therapy.

<Outline of gene detection carrier>
The gene detection carrier of the present invention can be prepared by immobilizing the predetermined polynucleotide on a substrate such as a substrate, a porous body, a microtiter plate, or a bead.

  The material, size, shape, etc. of the substrate on which the polynucleotide is to be immobilized are not particularly limited, and any substrate capable of immobilizing the polynucleotide can be used.

  Examples of the material of the substrate include inorganic materials such as silicon, glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, silicon nitride, and magnetic materials, polyethylene, polypropylene, polyisobutylene, polyethylene terephthalate, Saturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine Resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene polymer, silicone resin, polyphenylene oxide, polysulfone, nitrocellulose, nylon Emissions, polymethyl methacrylate, polyphenylene sulfone polyphenylene, polyethersulfone, polyether ketone, fluoroethylene copolymer, can be formed using an organic material such as polymethylpentene. Further, a composite of the above inorganic material and organic material can also be used.

  For example, the method disclosed in Science 251: 767-773 (1991) can be used as a method for immobilizing a polynucleotide on a substrate. In addition to this method, an improved method for immobilizing a polynucleotide on a substrate is known, and the polynucleotide may be immobilized using such an improved method.

  When a substrate made of an organic material or glass is used, the polynucleotide can be stably immobilized by coating the surface of the substrate with polylysine, aminosilane, or the like.

  In the present specification, “polynucleotide” means a substance in which two or more nucleosides are linked by a phosphate ester bond. “Nucleoside” includes, but is not limited to, deoxyribonucleoside and ribonucleoside.

  Furthermore, as the polynucleotide to be immobilized on the substrate of the present invention, oligonucleotides such as RNA, DNA, PNA, methyl phosphonate nucleic acid and S-oligo, and polynucleotides such as cDNA and cRNA can be used.

  In this specification, the “promoter region” does not refer only to a region directly involved in the transcription initiation reaction such as a TATA box, but affects the efficiency of the transcription initiation reaction by being present in the vicinity of the region or remotely. A sequence including a regulatory sequence that provides is also referred to. Therefore, it should be noted that the term “promoter region” includes only sequences involved in the transcription initiation reaction, only regulatory sequences, and sequences in which both are linked.

  “ISRE” means a base sequence consisting of about 12 to 15 nucleotides present in the transcriptional regulatory region of a gene induced by stimulation with interferon α, β, or γ.

  Examples of the polynucleotide immobilized on the substrate include the following (a) to (e).

  (A) a polynucleotide represented by any one of SEQ ID NOs: 1, 2, 3, 4;

  (B) A polynucleotide obtained by deleting, substituting, or adding one or several nucleotides excluding position 455 in the polynucleotide shown in (a).

  Examples of deletions include deletions at positions 128, 133, 152, 508, and 543, examples of substitutions are substitution at position 330 (G → T), substitution at position 542 (C → G) As an example of addition, addition to position 501 (C) can be mentioned.

  Furthermore, the polynucleotide of SEQ ID NOs: 1, 2, 3, 4 or a fragment of the polynucleotide is added to a promoter, an enhancer, an upstream activation sequence, a silencer, an upstream inhibitory sequence, an attenuator, a poly A tail, a nuclear translocation signal, Kozak sequence, ISRE, drug resistance factor, signal peptide gene, transmembrane region gene, luciferin, green fluorescent protein, phycocyanin, marker protein gene including horseradish peroxidase, interferon responsive protein gene, non-interferon responsive protein A modified polynucleotide in which at least one functional polynucleotide selected from the group consisting of these genes is linked may be used.

  In addition, among the nucleotide sequences of the polynucleotides shown in SEQ ID NOs: 1, 2, 3, and 4, the nucleotide involved in the effectiveness of interferon therapy is one nucleotide present at the SNP site located at position 455. The polynucleotide to be immobilized on the substrate may be a fragment of the polynucleotide containing the SNP site at position 455.

  When the fragment is used as a polynucleotide to be immobilized on a substrate, it is preferably 11 nucleotides or more and 30 nucleotides or less. If the length of the polynucleotide immobilized on the substrate is excessively long, it will be difficult to distinguish one nucleotide difference. On the other hand, if the length of the polynucleotide immobilized on the substrate is too short, it is difficult to determine the base sequence of the polynucleotide contained in the sample.

Particularly, suitable polynucleotides to be immobilized on the substrate are (c) fragments of the polynucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 containing the SNP site at position 455, A polynucleotide having the sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 corresponding to positions 441 to 456 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (that is, the ISRE) fragment.

  (D) A fragment of the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 containing the SNP site at position 455, corresponding to positions 449 to 459 of SEQ ID NO: 1 to SEQ ID NO: 4. A fragment comprising the polynucleotide of SEQ ID NOs: 9, 10, 11, 12. Particularly, since (d) includes a base sequence having a length approximately equal to the SNP and having the same length in the front and rear, the base sequence is determined with high accuracy. In order to perform detection with higher accuracy, a fragment containing a polynucleotide corresponding to positions 447 to 461 of SEQ ID NO: 1 to SEQ ID NO: 4 is desirable.

Furthermore, suitable polynucleotides to be immobilized on the substrate may be (e) complementary strands of the polynucleotides shown in (a) to (d) above.

  The complementary strands of the sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 (that is, the ISRE) are sequences represented by SEQ ID NOs: 13, 14, 15, and 16.

  The complementary strands of the sequences represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are the sequences represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.

  The gene detection carrier of the present invention uses the above-mentioned predetermined polynucleotide immobilized on a substrate as a probe, detects a hybridization reaction between this probe and a polynucleotide extracted from the subject, and a promoter region of the subject's human MxA gene Is used to determine which one has the SNP site.

<How to use the carrier for gene detection>
A method for determining the sequence of a polynucleotide specifically extracted from a subject using the gene detection carrier will be described below.

  There are two types of means for determining the sequence of a polynucleotide extracted from a subject using the gene detection carrier: (1) a method using a labeling substance and (2) an electrochemical method.

  First, the method using the labeling substance (1) will be specifically described.

  First, a sample containing a polynucleotide (body fluid such as blood, blood cell components, biopsy tissue, cultured cells, etc.) is collected from a subject such as a mammal including a human. Since the polynucleotide is widely contained in the body, it may be any sample collected from an individual. A preferred sample is blood.

  Subsequently, if necessary, separation and extraction operations such as phenol extraction and ethanol precipitation, and amplification operations such as PCR are performed, and then the sample polynucleotide contained in the sample is extracted. As a method for extracting the polynucleotide, for example, any extraction method other than phenol extraction and ethanol precipitation can be used. When mRNA is extracted, it may be applied to an oligo dT column. When the amount of the sample polynucleotide is small, an operation of amplifying the sample polynucleotide may be performed as necessary.

  Thereafter, a labeling operation for obtaining a labeled polynucleotide by applying a labeling substance to the sample polynucleotide is performed. Moreover, you may mix the secondary probe containing the polynucleotide which provided the labeling substance without labeling to sample polynucleotide.

  Examples of labeling substances include luminescent substances such as fluorescent substances, detectable substances such as haptens, enzymes, radioisotopes, and electrode active materials, but are not particularly limited. As for the label for the sample polynucleotide, it is desirable to label the promoter region. Various known techniques can be used to label the promoter region. However, PCR reactions using labeled primers may be useful because amplification and labeling can be performed simultaneously.

  Subsequently, the sample polynucleotide and the polynucleotide on the substrate are placed under hybridization reaction conditions. That is, first, the labeled sample polynucleotide is added to a reaction solution for hybridization, and then the reaction solution is brought into contact with the gene detection carrier of the present invention. If the sample polynucleotide has a complementary strand of nucleotides on the substrate, the sample polynucleotide undergoes a hybridization reaction with the polynucleotide on the substrate and is immobilized on the substrate.

  Hybridization reaction conditions may include a reaction temperature in the range of 10 to 90 ° C. and a reaction time of about 1 minute to overnight. During the reaction, the reaction rate can be increased by an operation such as stirring or shaking. The reaction solution for hybridization may be a buffer solution having an ionic strength in the range of 0.01 to 5 and a pH in the range of 5 to 10. Dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine breast DNA, EDTA, a surfactant and the like may be added to the reaction solution.

  Then, it wash | cleans suitably.

  Subsequently, in order to determine which test subject has the SNP site, the presence or absence of a hybridization reaction between the sample polynucleotide and the polynucleotide on the substrate is detected.

  Among the polynucleotides comprising any of the above (a) to (e) immobilized on a substrate, the sample polynucleotide is hybridized to a polynucleotide in which the SNP site is thymine or its complementary strand If detected, the SNP site of the polynucleotide comprising the promoter region of the human MxA gene of the subject is thymine. On the other hand, if it is detected that the sample polynucleotide is hybridized to the polynucleotide having the SNP site of guanine or its complementary strand, the SNP site of the polynucleotide containing the promoter region of the human MxA gene of the subject is guanine. is there. The same applies to adenine and cytosine.

  This detection is performed by detecting the labeled polynucleotide in the sample or the label in the secondary probe using an appropriate detection device corresponding to the type of label. When the label is a fluorescent substance, for example, the label may be detected using a fluorescence detector.

  As a carrier for gene detection, one of the polynucleotides (a) to (e) above, wherein the SNP site is thymine or its complementary strand, and guanine or its complementary on one substrate At least one selected from the group consisting of a strand, adenine or its complementary strand, cytosine or its complementary strand may be immobilized together, in which case the type of polynucleotide and the substrate It is necessary to specify in advance at least one of the position of each polynucleotide and the type of label. Depending on the position of the detected label and / or the type of the label, it becomes clear which polynucleotide on the substrate hybridized with the polynucleotide in the sample.

  In the method using the labeling substance of (1), the sequence of the sample polynucleotide can also be determined when a carrier for gene detection in which the sample polynucleotide collected from the subject is immobilized on a substrate is used. That is, a polynucleotide solution selected from (a) to (e) having a sequence previously given a different label and having a different kind of SNP base is brought into contact with the polynucleotide in the solution. The sample polynucleotide on the electrode is placed under hybridization reaction conditions. If the complementary strand of the sample polynucleotide on the substrate is present in the solution, the sample polynucleotide undergoes a hybridization reaction with the polynucleotide on the substrate and is immobilized on the substrate. Thereafter, it is possible to detect which sequence of the polynucleotide has undergone the hybridization reaction by detecting the type of the labeling substance, and to know the sequence of the sample polynucleotide.

  When the sample polynucleotide is hybridized with the polynucleotide having thymine at the SNP site or its complementary strand by the above method, the subject has thymine at the SNP site. Can be expected to be effective. In contrast, the polynucleotide in the sample hybridized with the polynucleotide having guanine at the SNP site or a complementary strand thereof, and did not hybridize with the polynucleotide having thymine at the SNP site or the complementary strand thereof. Sometimes, the subject does not have thymine at the SNP site and can therefore be predicted that interferon therapy is not effective.

  Next, the electrochemical method (2) will be specifically described.

  In the method using the labeling substance in (1) above, the presence or absence of a hybridization reaction between the sample polynucleotide and the polynucleotide on the substrate was detected by detecting the labeling substance. The presence or absence of a hybridization reaction is detected electrochemically.

  In the electrochemical method, a polynucleotide-immobilized electrode is used as a gene detection carrier. A polynucleotide-immobilized electrode (hereinafter referred to as an electrode of the present invention) is obtained by immobilizing any of the polynucleotides (a) to (e) on a substrate made of a conductive material.

  The preferred conductive material on which the polynucleotide is to be immobilized is gold, although other materials can be used. For example, gold alloys, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium, tungsten and other simple metals and alloys thereof, carbon such as graphite and glassy carbon, or oxides and compounds thereof Can be used. These conductive materials may be formed on another substrate by plating, printing, sputtering, vapor deposition, or the like.

  The method for immobilizing a polynucleotide on a substrate made of a conductive material is not particularly limited. For example, it can be easily performed by utilizing a bond between a thiol group introduced into a polynucleotide and gold. In addition, immobilization is possible by physical adsorption, chemical adsorption, hydrophobic bond, embedding, covalent bond, and the like. Further, a condensing agent such as a biotin-avidin bond or carbodiimide can also be used. In these cases, immobilization can be facilitated by modifying the surface of the conductive material with molecules having a functional group in advance. Furthermore, in order to suppress non-specific adsorption of the polynucleotide on the substrate surface, it is desirable to coat the substrate surface with a mercaptan such as mercaptoethanol or a lipid such as stearylamine.

  Hereinafter, as an example, a method for immobilizing a polynucleotide on a gold substrate will be described.

  First, the gold substrate is washed with deionized water and then activated. For activation, a 0.1 to 10 mmol / L sulfuric acid solution is used. In this solution, the potential is scanned in the range of −0.5 to 2 V (vs Ag / AgCl) and in the range of 1 v / s to 100,000 v / s. As a result, the surface of the gold substrate is activated to a state where the polynucleotide can be immobilized.

  A thiol group is introduced at the 5 'or 3' end of the polynucleotide to be immobilized. The thiolated polynucleotide is dissolved in a solution of a reducing agent such as DTT until immediately before immobilization, and DTT is removed by gel filtration or extraction with ethyl acetate immediately before use. For immobilization, the probe was dissolved in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 in a range of 1 ng / ml to 1 mg / mL. Immerse. The immobilization reaction is performed in the range of 4 to 100 ° C. for about 10 minutes to overnight. It is desirable that the substrate after immobilizing the polynucleotide is stored under conditions where no nuclease is present, preferably in the dark.

  When immobilizing a polynucleotide on a substrate, the polynucleotide can be immobilized relatively easily by using an immobilization device called a DNA spotter or DNA arrayer. At this time, it is desirable to use an ink jet type or electrostatic type spotter in order not to damage the surface of the substrate. It is also possible to synthesize polynucleotides directly on the substrate surface.

  The electrode of the present invention preferably constitutes a so-called DNA chip in which a conductor and the electrode of the present invention electrically connected to the conductor are disposed on a substrate. In the DNA chip, a conductive material such as a metal wire appropriately coated with an insulating material is disposed on a substrate, preferably an insulating substrate, and then the electrode on which the predetermined polynucleotide is immobilized in advance is electrically conductive. Created by connecting to the body so that it can be energized.

Each polynucleotide having a different sequence needs to be immobilized on a plurality of different electrodes insulated from each other. When a plurality of electrodes are installed on the substrate, the number of electrodes to be installed on the substrate may be practically 10 ¹ to 10 ⁵ .

  When a plurality of electrodes of the present invention are installed on a substrate, a scanning line may be further installed, and a switching element may be arranged at each intersection of the conductor and the scanning line. Accordingly, it is possible to quickly determine whether a hybridization reaction has occurred for each electrode by sequentially applying a voltage to each electrode.

  In the detection of the presence or absence of a hybridization reaction using the electrode of the present invention, in addition to the electrode of the present invention, at least a counter electrode and, if necessary, a reference electrode as appropriate, as in other general electrochemical detection methods. Deploy. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode can be used. These electrodes are preferably disposed on the same substrate as the electrode of the present invention.

  In order to determine the sequence of a polynucleotide collected from a subject using the electrode of the present invention, the following operation is performed.

  First, a sample containing a nucleic acid is collected from a subject such as a mammal including a human, and the above-described extraction operation, amplification operation, and labeling as necessary are performed. These operations are the same as the method (1). However, labeling is not always necessary.

  Next, the solution containing the sample is brought into contact with the electrode of the present invention, and the sample polynucleotide and the polynucleotide on the electrode are placed under hybridization reaction conditions. If the sample polynucleotide has a complementary strand of nucleotides on the substrate, the sample polynucleotide undergoes a hybridization reaction with the polynucleotide on the substrate and is immobilized on the substrate. The operation and conditions are the same as in the method (1). Thereafter, the electrode of the present invention is washed.

  Furthermore, by adding a solution of a conductive double-stranded recognizer in the vicinity of the electrode of the present invention, the double-stranded polynucleotide formed by hybridization of the polynucleotide immobilized on the electrode and the sample polynucleotide is hybridized. The polynucleotide is intercalated with a double-stranded recognizer. The detection of the presence or absence of a hybridization reaction between the sample polynucleotide and the polynucleotide on the substrate is performed by detecting a double-stranded intercalated double-stranded polynucleotide on the electrode when a voltage is applied to the electrode. This is done by detecting an electrical signal generated from the recognizer.

  The double-stranded recognizer used here is not particularly limited. For example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisacridine, trisintercalator, polyintercalator Etc. can be used. A metal complex called a metallointercalator such as ruthenium, cobalt, or iron, or an organic compound such as ethidium bromide can be used. In addition to these, it is also possible to use a biopolymer such as Hoechst 33258 or a groove binder such as a cyanine dye, an antibody, or an enzyme.

  The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used.

  After reacting the electrode of the present invention with a double-stranded recognizer, the electrode is further washed as necessary. Furthermore, the electrode of the present invention is used as a working electrode, an electric potential is applied between the working electrode and a separately provided counter electrode, and an electrochemical signal is detected by measuring a current between both electrodes. The detection of the electrochemical signal may be performed with such two electrodes (working electrode and counter electrode), or may be performed with three electrodes using the working electrode, the counter electrode, and the reference electrode. In the detection of an electrochemical signal, the potential can be swept at a constant speed, applied in pulses, or a constant potential can be applied. For the measurement, current and voltage are controlled by using a potentiostat, a digital multimeter, a function generator or the like. Based on the obtained current value, the concentration of the target gene is calculated from the calibration curve.

  Among the polynucleotides (a) to (e), the SNP site is thymine or its complementary strand, the SNP site is guanine or its complementary strand, and the SNP site is adenine. One or its complementary strand, the one whose SNP site is cytosine, or its complementary strand constitutes a DNA chip fixed on different electrodes insulated from each other and disposed on the same substrate. Is desirable for performing highly accurate measurements. In that case, an electrochemical signal corresponding to each polynucleotide is detected from each electrode.

  The sequence of the sample polynucleotide can also be determined using the electrochemical method of (2) and using an electrode in which the sample polynucleotide collected from the subject is immobilized on the electrode. That is, by contacting a polynucleotide solution of any one of (a) to (e) whose sequence is known in advance, the polynucleotide in the solution and the sample polynucleotide on the electrode are placed under hybridization reaction conditions. . If the complementary strand of the sample polynucleotide on the substrate is present in the solution, the sample polynucleotide undergoes a hybridization reaction with the polynucleotide on the substrate and is immobilized on the substrate. By detecting the hybridization reaction, the sequence of the sample polynucleotide can be known.

  When the hybridization reaction between the sample polynucleotide and the polynucleotide having thymine at the SNP site or its complementary strand is detected by the above method, the subject has thymine at the SNP site, Therefore, it can be predicted that interferon therapy is effective. In contrast, a hybridization reaction between a polynucleotide in a sample and a polynucleotide having a component other than thymine at the SNP site or a complementary strand thereof is detected, and a polynucleotide having a thymine at the SNP site or a complementary strand thereof When no hybridizing reaction is detected, the subject does not have thymine at the SNP site and can therefore be predicted that interferon therapy is not effective.

<Electrical detection device>
An example of a gene detection apparatus for determining the sequence of a polynucleotide extracted from a subject using the electrode of the present invention is shown below. That is, a reaction part for performing a hybridization reaction between the electrode of the present invention and a polynucleotide immobilized on the electrode, application means for applying a voltage to the nucleic acid immobilized on the electrode, A gene detection apparatus comprising measurement means for measuring a signal generated by operation of an application means.

<Basic configuration of electrical detection device>
FIG. 1 shows a basic embodiment of such a gene detection apparatus.

  In FIG. 1, 11 is an electrode on which a polynucleotide 12 is immobilized. Reference numeral 13 denotes a reaction tank, and a power source 14 is connected to form a circuit via the electrode 11 and the reaction tank 13. In addition, an ammeter 15, a voltmeter 16, and a variable resistor 17 are arranged inside the circuit, and after conducting a hybridization reaction in the reaction vessel 13, a current is applied to the circuit by applying a voltage. It is configured to measure.

  In order to perform the test using the gene detection apparatus of FIG. 1, a buffer solution and a polynucleotide-containing sample collected from a subject are held in the reaction tank 13, and the sample polynucleotide and the polynucleotide 12 on the electrode 11 are retained. Hybridization reaction is performed between the two. Conditions such as hybridization temperature and time are as described above. Subsequently, the reaction vessel 13 and the electrode 11 are preferably washed, the hybridization solution containing the intercalating agent is filled in the reaction vessel 13 and the power source 14 is operated, and the power source value or voltage value is measured using the ammeter 15 or the voltmeter 16. Measure etc.

  FIG. 1 shows the most basic embodiment of the gene detection apparatus of the present invention. However, in order to fully automate the test, the gene detection apparatus of the present invention is further complicated as shown in FIG. You may take a simple structure.

<Configuration of automated electrical detection device>
2 includes an electrode holder 21 for holding a polynucleotide-immobilized electrode, a reaction tank 22, a first washing tank 23, an intercalating agent reaction tank 24, a second washing tank 25, and electrochemical measurement. A tank 26, a reaction tank 22, a first cleaning tank 23, an intercalating agent reaction tank 24, a second cleaning tank 25, a moving device 27 on which an electrochemical measurement tank 26 is placed, an analysis unit 28, and an operation unit 29 are provided. is doing. The movement of the electrode between these tanks is achieved by moving the reaction tank or the like using the moving device 27.

<Usage of automated electrical detection device>
The usage of the detection apparatus 20 is as follows.

  First, a sample or a solution containing a polynucleotide previously extracted from the sample is placed in the reaction tank 22, and the electrode fixed to the electrode fixing holder 21 is immersed in the reaction tank 22. Next, after performing a hybridization reaction in the reaction tank 22, the electrode fixing holder 21 is moved to the first washing tank 23. In the 1st washing tank 23, it wash | cleans and removes the nucleic acid, protein, etc. which were couple | bonded with the electrode nonspecifically. After washing, the electrode fixing holder 21 is transferred to the intercalating agent reaction tank 24, and the intercalating agent is intercalated into the double-stranded polynucleotide formed by the hybridization reaction. Subsequently, the electrode fixing holder 21 is moved to the second cleaning tank 25 and cleaned, and then the electrode fixing holder 21 is transferred to the electrochemical measurement tank 26 to perform electrochemical measurement. After completion of the measurement, the concentration of the polynucleotide in the sample is output with reference to a calibration curve recorded in advance in the analysis unit 28. The above operation is performed through the operation unit 29 electrically connected to each tank and unit.

  The present invention provides a kit comprising the carrier for gene detection of the present invention and a buffer. Thus, providing the gene detection carrier of the present invention and the buffer as a set is convenient because the test can be performed immediately.

  The buffer to be used in the kit of the present invention can be any buffer used in the hybridization reaction. For example, phosphate buffer, carbonate buffer, Tris buffer and the like can be mentioned, but are not limited thereto.

  The kit may include a primer for gene amplification, a fluorescent dye-labeled nucleotide, an enzyme, a purification column, and the like together with or in place of the buffer solution.

  The present invention provides a kit comprising the electrode of the present invention and a double-stranded recognizer.

  The double-stranded recognizer to be used in the kit of the present invention can be, for example, the double-stranded recognizer described above for the electrode of the present invention.

  A kit, a primer for gene amplification, an enzyme, a buffer, nucleotides, or the like may be attached to the kit together with or in place of the double-stranded recognizer.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1:
In this example, a gene (hereinafter referred to as MxA (T)) in which the nucleotide at position −88 of the promoter region of the MxA gene (corresponding to position 455 in the nucleotide sequence of SEQ ID NO: 1) is thymine is heterozygous or homozygous. It has been demonstrated that HCV-infected persons having a zygote having a zygote have a better response to interferon therapy than HCV-infected persons having a gene whose nucleotide is guanine (hereinafter referred to as MxA (G)).

<Subject>
115 patients with histologically proven chronic hepatitis C who were on interferon therapy and 42 healthy people who were anti-HCV negative participated in the study. These people are all Japanese and are not related to each other.

  Of the 115 patients, 52 were persistent with serum alanine aminotransferase levels in the normal range and always negative for HCV RNA during the follow-up period of at least 6 months after termination of interferon therapy. Responders (hereinafter referred to as SR), 63 patients were non-responders who remained positive for HCV RNA or relapsed hepatitis C during the follow-up period, regardless of ALT level (Hereinafter referred to as NR). All patients received interferon alpha and / or beta over a total dose of 3 million units.

<MxA gene analysis>
Nucleic acids were extracted from PBMC collected from patients and healthy controls. The nucleic acid was subjected to PCR to amplify 599 nucleotide DNA including the promoter region of the MxA gene.

  The outline of PCR is as follows.

  0.05 μg of nucleic acid was added to Taq-Gold (Perkin Elmer), oligonucleotide primer # MXAF01 (forward primer, positions −569 to −540) of SEQ ID NO: 12, and oligonucleotide primer # MXAF02 (reverse primer, + 30- + 1 position) and then reacted under the cycle conditions of [95 ° C. for 10 minutes], [95 ° C. for 10 seconds, 68 ° C. for 60 seconds] × 55, [72 ° C. for 7 minutes]. Twelve of 157 samples were sequenced by directly decoding the PCR product.

  After identifying SNP sites in the amplified region by sequencing, an RFLP system was constructed to detect allele nucleotides distinguishably. The 599 nucleotide PCR product of all patients was digested with HhaI (GCG ↓ C) and electrophoresed in an agarose gel to determine whether either or both of the 482 nucleotide band and the 533 nucleotide band were generated. .

<Statistical analysis>
Group data were compared by Fisher's exact test with or without Yates correction. p &LT; 0.05 was considered statistically significant.

<Result>
By sequencing 12 samples, polymorphic sites were identified in the promoter region of the MxA gene. SNP was present at position -88 of the promoter region (G and T), and this SNP was contained in a region similar to ISRE shown in FIG.

  In the subsequent RFLP by HhaI, the genotype of the MxA gene was determined for all 157 samples. In the assay, samples with guanine at the polymorphic site showed a 482 nucleotide bunt on the electrophoresis gel. In contrast, when guanine was replaced with thymine, the restriction site recognized by HhaI disappeared, indicating a band of 533 nucleotides. When the promoter region whose polymorphic site is guanine and the promoter region whose polymorphic site is thymine are heterozygous, both a 482 nucleotide band and a 533 nucleotide band are detected (see FIG. 4).

  As shown in Table 1, 62% of patients in the NR group had a homozygous promoter region in which the polymorphic site was guanine (GG homo), but in the SR group, 31% of patients were GG homozygous (p = 0.0009; SR vs NR). Conversely, in the NR group, 35% of patients had a promoter region in which the polymorphic site is guanine and a promoter region in which the polymorphic site is thymine in a heterozygote (GT hetero), In the SR group, 60% of patients were GT heterozygous (p = 0.0082; SR vs. NR). T and T homozygous patients were 3.2% in the NR group and 10% in the SR group, respectively (p = 0.2956; SR vs NR).

The frequency of alleles having a promoter region in which the polymorphic site is guanine was 0.606 in the SR group, and 0.794 in the NR group (p = 0.0018; SR vsNR).

Furthermore, the above results that the number of GG homozygous individuals was significantly less in the SR group than in the NR group were also found to be independent of the genotype of the HCV that the patient was infected with.

  As is clear from Table 2, both the group of patients infected with HCV genotype 1b (HCV 1b group) and the group of patients infected with HCV genotype 2a or 2b (HCV 2a / 2b group) In the NR group, 62% were G and G homozygous individuals, whereas in the SR group, 28% and 32% were G and G homozygous individuals, respectively. The results shown in Table 2 revealed that the SR group had significantly fewer G and G homozygous individuals regardless of the genotype of the HCV infected by the patient (HCV 1b group; p = 0.0321, HCV 2a / 2b group; p = 0.0318).

  As described above, according to this example, an HCV-infected person having an MxA promoter region whose polymorphic site is thymine in a heterozygote or homozygote is highly responsive to interferon therapy regardless of the genotype of the infected HCV. It was demonstrated to show.

  In addition, this example also suggests that HCV-infected persons whose MxA promoter region where the polymorphic site is not guanine are heterozygous or homozygous show high responsiveness to interferon therapy.

  Furthermore, since the polymorphic site is present in ISRE, it can be said that these also apply to diseases other than hepatitis C.

Example 2:
As revealed in Example 1, HCV-infected persons whose MxA promoter SNP is T type have a high therapeutic effect on hepatitis due to interferon administration. On the other hand, when the SNP is type G, the therapeutic effect is low. The reason for this is that T-type, which is the base sequence for ISRE function, responds correctly to interferon stimulation, but G-type differs from that sequence by only one base, so it does not respond to interferon stimulation and does not respond to MxA protein. It is interpreted that the production amount is low.

  In view of this situation, patients with HCV hepatitis whose MxA promoter SNPs are type C and type A do not respond to the interferon MxA promoter as in type G, and it is estimated that the therapeutic effect is low.

  To prove these things, a plasmid having a luciferase gene downstream of the MxA promoter was constructed and transfected into human cells (HeLa cells and ovarian cancer cells). And about each of the MxA promoter which has 4 types of SNP (T type, G type, A type, C type), these investigated the luciferase activity produced as a result of responding to interferon.

  The results are shown in FIGS. FIG. 5 is an example induced in Hela cells using interferon α, FIG. 6 is an example induced in ovarian cancer cells using interferon α, and FIG. 7 is induced in Hela cells using interferon β. FIG. 8 shows an example of induction in ovarian cancer cells using interferon β. In these figures, + indicates luciferase activity when interferon is added, and-indicates luciferase activity when no interferon is added. All of these results are average values of three experiments, and the standard deviation is indicated by a bar.

  As is clear from the figure, the T-type MxA promoter shows the highest value in all cases, and patients with HCV hepatitis having S-types of type A and type C have interferon α and interferon β, as in type G. Response to is low. Therefore, it is predicted that the therapeutic effect by interferon is low.

Example 3:
In this example, an operation for predicting the effectiveness of interferon therapy using the carrier for gene detection of the present invention will be described with reference to FIG.

  First, in order to prepare the carrier for gene detection of the present invention, using the primers of SEQ ID NO: 21 and SEQ ID NO: 22, the polynucleotide fragment of SEQ ID NO: 1, including the SNP site, the SNP site is thymine The T-type fragment 31 and the G-type fragment 32 containing the SNP site of the polynucleotide of SEQ ID NO: 2 and having the SNP site being guanine were amplified and prepared with distilled water so as to be 2 μg / μL.

  Next, an equal amount of 4 mg / mL nitrocellulose (DMSO solution) was added to each fragment, and after heat denaturation, spotted on a slide glass 33 coated with polylysine. After the spots were sufficiently dried, an immobilization treatment by ultraviolet irradiation was performed to obtain a gene detection carrier on which the T-type fragment 31 and the G-type fragment 32 were immobilized.

  Next, the sample polynucleotide extracted from the blood of the subject was amplified with the primers of SEQ ID NO: 20 and SEQ ID NO: 21, and then FITC-labeled with an ECL labeling system manufactured by Pharmacia. The reaction was carried out with the support for 12 hours at 65 ° C. After the reaction, the signal was measured with a fluorescence detector.

  As a result, a fluorescent signal was observed from the spot where the T-type fragment was immobilized, and it was found that the SNP of the sample polynucleotide used in this test was T-type.

Example 4:
In this example, a test using a gene detection carrier in which a fragment in a sample is immobilized will be described with reference to FIG.

  Fragments (sample A: 41, sample B: 42) obtained by amplifying the polynucleotide detected from the subject using the primers of SEQ ID NO: 21 and SEQ ID NO: 22 were prepared with distilled water so as to be 2 μg / μL. Next, after adding an equal amount of 4 mg / L nitrocellulose (DMSO solution), it was heat-denatured and spotted on a slide glass 43 coated with polylysine. After the spots were sufficiently dried, an immobilization treatment by ultraviolet irradiation was performed.

  A polynucleotide fragment of SEQ ID NO: 1 that has been fluorescently labeled in advance and is a T-type fragment (JOE label) that includes the SNP site and is thymine, and a polynucleotide fragment of SEQ ID NO: 2 that has been fluorescently labeled A G-type fragment (5-FAM label) containing the SNP site and containing the SNP site, and a polynucleotide fragment of SEQ ID NO: 3, which has been fluorescently labeled in advance, and comprising the SNP site and the SNP site is adenine A type A fragment (TAMRA label), a previously fluorescently labeled polynucleotide fragment of SEQ ID NO: 4, comprising the SNP site and a C type fragment (ROX label) in which the SNP site is cytosine were prepared in advance. The DNA chip was reacted at 50 ° C. for 1 hour.

  After the reaction, the signal was measured with a fluorescence detector. As a result, only a signal derived from the fluorescence of HEX was obtained, and it was found that the SNP of the sample polynucleotide was only T type.

Example 5:
In this example, a gene detection carrier using electrochemical detection will be described with reference to FIG.

  Using the primers of SEQ ID NO: 21 and SEQ ID NO: 22, the T-type fragment 51 and G-type fragment 52 of SEQ ID NO: 1 were amplified and prepared with distilled water to 2 μg / μL. Next, 2-hydroxyethyl disulfide and 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide were reacted to introduce a thiol group at the end of the amplified product. It was spotted on a gold electrode 53 prepared in advance and reacted for 1 hour so as not to dry. The sample was amplified using the primers of SEQ ID NO: 21 and SEQ ID NO: 22, heat-denatured, and reacted with a DNA chip prepared in advance at 65 ° C. for 1 hour. After the reaction, voltammetry was performed in a 10 μmol / L Hoechst 33258 solution, and the oxidation current value of Hoechst 33258 was measured. As a result, a significant signal was obtained only from the electrode on which the G-type fragment was immobilized, and this sample was found to be G-type.

FIG. 1 is a diagram showing an example of a gene detection apparatus according to an embodiment of the present invention. FIG. 2 is a diagram illustrating an example of an automated electron detection apparatus according to an embodiment of the present invention. FIG. 3 is a diagram showing the base sequence of the promoter region of the MxA gene. FIG. 4 is a photograph showing a result of electrophoresis of RFLP using HhaI according to the example. FIG. 5 shows the responsiveness to interferon α in Hela cells for each of the MxA promoters having four types of SNPs (T type, G type, A type, and C type), and the luciferase activity under the control of the promoter. It is a graph which shows the result compared as a parameter | index. FIG. 6 shows the responsiveness to interferon α in ovarian cancer cells for each of the MxA promoters having four types of SNPs (T type, G type, A type, and C type), and the luciferase activity under the control of the promoter. It is a graph which shows the result compared as a parameter | index. FIG. 7 shows responsiveness to interferon β in Hela cells for each of the MxA promoters having four types of SNPs (T type, G type, A type, and C type), and the luciferase activity under the control of the promoter. It is a graph which shows the result compared as a parameter | index. FIG. 8 shows the responsiveness to interferon β in ovarian cancer cells for each of the MxA promoters having four types of SNPs (T type, G type, A type, and C type), and the luciferase activity under the control of the promoter. It is a graph which shows the result compared as a parameter | index. FIG. 9 is a diagram showing a gene detection carrier according to one embodiment of the present invention. FIG. 10 is a diagram showing a gene detection carrier according to another embodiment of the present invention. FIG. 11 is a diagram showing a gene detection carrier according to another embodiment of the present invention.

Explanation of symbols

  11. Electrode 12. Polynucleotide 13. Reaction tank 14. Power supply 15. Ammeter 16. Voltmeter 17. Variable resistance 20. Detection device 21. Electrode holder 22. Reaction tank 23. First washing tank 24. Intercalator reaction tank 25. Second washing tank 26. Electrochemical measurement tank 27. Mobile device 28. Analysis unit 29. Operation unit 31. T-fragment 32. G-type fragment 33. Glass slide 41. Sample A 42. Sample B 43. Glass slide 51. T-fragment 52. G-type fragment 53. Gold electrode

Claims (5)

A method for predicting the effectiveness of interferon therapy from a sample derived from an individual to be administered interferon, comprising:
(1) for a sample derived from the individual, determining the genotype at position -88 of the MxA promoter of the individual;
(2) Predicting the effectiveness of interferon therapy from the type determined in (1),
A method for predicting the effectiveness of interferon therapy for a sample from an individual to be administered interferon comprising: A method of collecting data for predicting whether interferon therapy is effective from a sample derived from an individual infected with hepatitis C virus,
  Determining whether the sequence of the polynucleotide comprising the interferon responsive element in the sample comprises a portion that matches SEQ ID NO: 1. A method of collecting data for predicting whether interferon therapy is effective from a sample derived from an individual infected with hepatitis C virus,
  Determining whether the sequence of the polynucleotide comprising the interferon responsive element in the sample contains a portion that matches SEQ ID NO: 13. A method of collecting data for predicting whether interferon therapy is effective from a sample derived from an individual infected with hepatitis C virus,
  Determining whether the sequence of the polynucleotide comprising the interferon responsive element in the sample comprises a portion that matches SEQ ID NO: 9. A method of collecting data for predicting whether interferon therapy is effective from a sample derived from an individual infected with hepatitis C virus,
  Determining whether the sequence of the polynucleotide comprising the interferon responsive element in the sample comprises a portion that matches SEQ ID NO: 17.

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