JPWO2004035829A1 - Gene detection probe and electrochemical gene detection method - Google Patents

Abstract

A gene detection probe containing a redox unit at the end and capable of taking a hairpin structure is immobilized on an electrode and used as a DNA chip, so that the target gene can be easily and at a high S / N ratio without labeling the sample. Perform detection. We have developed a system that detects changes from the hairpin structure to the double-stranded structure associated with target gene recognition using changes in the electrochemical system.

Description

  The present invention relates to a hairpin probe that enables electrochemical nucleic acid detection, a target gene detection chip on which the hairpin probe is immobilized, and an electrochemical nucleic acid detection method using the same.

Various methods are used as gene analysis methods based on hybridization. In Southern hybridization or Northern hybridization, DNA or RNA separated by gel electrophoresis is transferred to a nylon membrane or the like and hybridized with a labeled probe. In the DNA chip or DNA microarray method, a group of DNAs whose sequences are known are fixed on a substrate such as glass in a densely aligned state, and these are used as probes to increase the DNA or RNA to be examined. Hybridization enables high-throughput gene analysis. In these analyzes by hybridization, it is common to label either a probe or a nucleic acid to be examined with a radioactive compound or a fluorescent substance. In the case of labeling with radioactive compounds, operation within the controlled area is premised, and the facilities that can be used are limited. On the other hand, the labeling with a fluorescent substance can be operated in a general laboratory, but uses a laser beam for detection, so that there is a problem that the detection device becomes large and expensive.
In a DNA chip or a DNA microarray, an electrochemical detection method is used as a detection means instead of fluorescence measurement. For example, the method disclosed in Japanese Patent No. 2573443 uses a nucleic acid probe immobilized on the electrode surface, a double-stranded nucleic acid that binds specifically to a double-stranded nucleic acid and is electrochemically active ( An insertion agent) is added to the reaction system, and a redox current derived from the insertion agent bound to a double-stranded nucleic acid comprising a nucleic acid probe and a target gene is measured. In this method, it is not necessary to label the nucleic acid molecule to be examined in advance, but operations such as addition of an intercalating agent to the measurement system and removal of an excessive intercalating agent are required following hybridization. Also, with such a method, the amount of intercalating agent binding to the nucleic acid molecule cannot be strictly controlled in the measurement system. Furthermore, it is considered difficult to perform high-level control of the measurement system, such as using different insertion agents for each probe on one electrode.

The present invention is simple and does not require special procedures (labeling of test target nucleic acid, addition of intercalating agent, addition of enzyme, etc.) in addition to adding the test target nucleic acid to the gene detection probe fixed to the electrode. It is another object of the present invention to provide a technique having a good S / N ratio. Another object of the present invention is to provide a technique capable of fixing a plurality of types of gene detection probes on a single electrode and simultaneously detecting these target genes.
In order to solve the above problems, in the present invention, a change from a hairpin structure (closed structure) to a double-stranded structure (open structure) associated with recognition of a target gene was positively utilized. That is, a DNA molecule containing reverse repeat sequences complementary to each other in the molecule and a target gene recognition sequence sandwiched between them was used. In the situation where the target gene does not exist, the DNA molecule forms a stable intramolecular double strand by a complementary inverted repeat sequence, and has a hairpin-like closed structure as a whole molecule. When the target gene is present, the target gene is recognized by the recognition sequence located in the loop in the hairpin structure, forms a double strand with each other, and becomes an open structure. In this process, the higher-order structure of the DNA molecule greatly changes from a single molecule in the hairpin state to a double strand (hairpin collapse) with the target gene.
Furthermore, a system has been developed in which a redox unit is bonded to one end of the DNA molecule to detect the decay state of the hairpin using an electrochemical reaction. For example, when a redox unit is bound to one end of a probe composed of a single-stranded nucleic acid having the above hairpin structure, the other end is fixed to an electrode, and this is hybridized with a target gene, the hairpin structure Collapses and the electrochemical system on the electrode changes. This change is due to the fact that the probe fixed to the electrode takes a hairpin structure in the situation where the target gene does not exist, so that the redox unit bound to the end is close to the electrode surface, but the target gene is recognized. When the double strand is formed, it is considered that the hairpin structure is opened and the redox unit is moved away from the electrode surface. This change can be measured as a change in an electrical signal such as a current to confirm the presence of the target gene.
In this way, by coupling the redox unit to the end of the probe that undergoes the largest structural change upon recognition of the target gene, the distance between the redox unit and the electrode is changed, thereby changing the redox unit to the redox unit. A technique for changing the derived electrical response was developed and the present invention was completed.
That is, the present invention is as follows.
(1) A probe for detecting a target gene comprising a linear molecule comprising a recognition sequence capable of recognizing a target gene and comprising at least one of a nucleic acid and a nucleic acid analog, and a redox unit,
Independently, it comprises a double-stranded stem and a single-stranded loop, the single-stranded loop forms a hairpin structure closed by the double-stranded stem, and the detected sequence indicating the presence of the target gene and the recognition sequence Forming a double strand, the hairpin structure is opened, and a portion of the probe has a sequence necessary to form a double-stranded portion with a detected sequence, and the oxidation The reducing unit is a probe for detecting a target gene, wherein when the hairpin structure is formed, the reducing unit is bound to one of two strands that are bound or adjacent to each other except the single-stranded loop.
(2) The sequence capable of forming the hairpin structure is composed of two inverted repeat sequences complementary to each other, and a target gene recognition sequence comprising a sequence complementary to the detected sequence provided between these repeat sequences The probe for detecting a target gene according to the above item (1), comprising:
(3) The probe for detecting a target gene according to (1) or (2) above, wherein at least a part of the single-stranded molecule is DNA or RNA.
(4) The gene detection probe according to (1) or (2) above, wherein at least a part of the single-stranded molecule is a peptide nucleic acid.
(5) The gene detection probe according to any one of (1) to (4) above, wherein the redox unit is contained at any one of the following positions a) and b).
a) One of the two strands constituting the double-stranded stem.
b) One end of the linear molecule.
(6) The gene detection probe according to any one of (1) to (6), wherein the redox unit is a quinone compound, a metallocene compound, a flavin compound, a porphyrin compound, a metal, or a metal compound.
(7) A chip for detecting a target gene comprising a carrier having an electrode surface and the probe for detecting a target gene according to any one of (1) to (6) above bound to the electrode surface. ,
When the hairpin structure is formed, the end region of the strand that is not bound to the reducing unit of the two strands that are bound or adjacent to each other other than the single-stranded loop is bound to the electrode. A chip for detecting a target gene.
(8) A plurality of different probes capable of specifying each of a plurality of different target genes is fixed to the electrode surface for each probe, and redox units having different electrical responses for the plurality of target genes The chip for detecting a target gene according to (7), which is bound to each probe.
(9) In a method for detecting a target gene using a probe,
Forming a reaction system on the electrode surface of the chip for detecting a target gene according to (7) above, in which the probe fixed on the electrode surface can form the hairpin structure;
In the reaction system, when the sample and the probe contain a sequence to be detected, a double strand is formed by hybridization of the probe and the sequence to be detected. A step of reacting under the condition that the hairpin structure of can be opened and converted into a single strand;
A method of detecting a target gene, comprising: applying a potential to the electrode of the chip that has undergone the reaction, and determining a state of hybridization between the probe and the sequence to be detected based on the obtained current value .
(10) The method for detecting a target gene according to (9), wherein a plurality of probes that recognize a plurality of different target genes are fixed to different electrodes for each probe.
(11) In a method for detecting a target gene using a probe,
Forming a reaction system on the electrode surface of the chip for target gene detection described in (8) above, in which the probe fixed on the electrode surface can form the hairpin structure;
In the reaction system, when the sample and the probe contain at least one kind of detected sequences corresponding to a plurality of different target genes, the probe and the detected sequence are high. Forming a double strand by hybridization, and reacting under a condition in which the hairpin structure of the probe is opened and converted into a single strand having no double-stranded portion;
Applying the potential to the electrode of the chip that has undergone the reaction, and determining the state of hybridization between the probe and the sequence to be detected based on the obtained current value. Detection method.
The above-mentioned hybridization state usually indicates whether the hairpin structure is broken by forming a double strand or whether the hairpin structure remains.
By using the gene detection probe and electrochemical gene detection method of the present invention, the target gene can be detected easily and quickly without any special operation other than preparing a nucleic acid as a sample. It becomes possible. In the present invention, since the higher order structure of the gene detection probe itself changes depending on the presence or absence of the target gene, the detection S / N ratio is large, and a highly reliable detection result can be provided. Therefore, it is effective for detecting a very small amount of genes, detecting SNPs, and the like.
In addition, by using a redox unit having different electrical responsiveness for each probe having a different target gene, a DNA chip in which these probes are fixed to a single electrode can be constructed. By using the DNA chip, simultaneous detection of a plurality of genes can be performed easily and at low cost.
The technique of the present invention does not require any special treatment except that the end of a DNA molecule capable of taking a hairpin structure is modified with a redox unit, and is extremely simple. By setting the conditions for hybridization between the probe and the target gene, it is possible to detect a slight sequence difference or the like of the target gene with high sensitivity.
In addition, since the redox unit can be changed depending on the type of sequence, a plurality of target genes can be obtained by immobilizing probes consisting of various sequences linked with units having different redox potentials on one electrode. It can be easily detected.

FIG. 1 is a schematic diagram of a typical gene detection probe in the present invention.
FIG. 2 is a diagram showing a typical gene detection flow in the present invention.
FIG. 3 is a schematic diagram of a single electrode / multiple detection type DNA chip using the gene detection probe of the present invention. The probe 1 has a redox unit 1 and the probe 2 has a redox unit 2.
FIG. 4 is a synthesis flow diagram of an anthraquinone modified uridine nucleotide.
FIG. 5 shows a differential pulse voltammogram when ODN2 (WT) is used as a specimen (when it has a sequence that perfectly matches the recognition sequence of the target gene of ODN1).
FIG. 6 shows a differential pulse voltammogram when ODN3 (G178MU) is used as a specimen (when it consists of a sequence that does not match the recognition sequence of the target gene of ODN1).
FIG. 7 shows a differential pulse voltammogram when ODN4 (F508) is used as a specimen (when it consists of a sequence that does not match the recognition sequence of the target gene of ODN1).
FIG. 8 is a graph in which the amount of current in differential pulse voltamography is relativized for three specimens of WT, G178MU, and F508.
FIG. 9 is a graph in which peak current values in differential pulse voltamgraphy are relativized for three samples of WT, G178MU, and F508.
FIG. 10 is a diagram showing a production process of ODN5, DMSO indicates dimethyl sulfoxide, and DTT indicates dithiothreitol.
FIG. 11 shows the DPV result when the probe ODN5 is used and ODN2 (WT) is used as a sample.
FIG. 12 shows a DPV result when the probe ODN5 is used and ODN3 (G178MU) is used as a sample.
FIG. 13 shows the DPV result when the probe ODN5 is used and ODN4 (F508) is used as a sample.
FIG. 14 shows the calculation result of the S / N ratio.
FIG. 15 shows the results of current values when 10 cycles in Example 4 were repeated.

Hereinafter, the present invention will be described in detail.
The “target gene detection probe” in the present invention refers to a molecule for detecting the presence in a sample of a nucleic acid molecule having a sequence for specifying a target gene as a detection target based on a hybridization reaction. . The gene detection probe in the present invention is composed of a linear molecule containing a redox unit, and has a closed hairpin structure consisting of a double-stranded stem and a single-stranded loop alone, but has a target gene. Under the conditions, the target gene has a sequence characteristic such that an open structure is formed by forming a double strand with the target gene. Examples of the structure capable of forming such a hairpin structure include, for example, those that each include a reverse repeat sequence complementary to each other and a sequence capable of recognizing a target gene between the repeat sequences. it can.
“Linear molecules” include nucleic acids (DNA or RNA) such as oligonucleotides, nucleic acid analogues (modified nucleic acids) consisting of chemically modified nucleotide units, and so-called textures containing DNA and RNA. Rick nucleotides, peptide nucleic acids (PNA), and other artificial linear molecules that can behave like nucleic acid molecules in hybridization reactions are included. The linear nucleic acid can be synthesized by, for example, a DNA synthesizer, and the synthesized nucleic acid may be amplified by PCR if necessary. Molecules similar to linear nucleic acids can be prepared by methods known per se.
“Double-stranded stem” refers to the double strand formed in the molecule, and “single-stranded loop” refers to the single-stranded portion that has become a loop as the double-stranded stem is formed. Say. “Closed structure” refers to a hairpin-like structure composed of a double-stranded stem and a single-stranded loop, and “open structure” refers to a state where the double-stranded stem is dissociated. The “single-stranded loop” does not necessarily have a loop shape and can take a random structure, but does not contribute to stabilization of the “closed structure”.
“Complementary inverted repeat sequence” is any sequence that is capable of forming a stable duplex within a molecule by Watson-Crick base pairing and is not related to the target gene. The repeating unit is preferably 3 bases or more, and more preferably 5 to 8 bases, but longer repeating units are suitable for some applications. Further, it is preferable that the repeating unit is arranged at both ends of the gene detection probe molecule or in the vicinity of the ends.
The “recognition sequence capable of recognizing the target gene” or the recognition sequence of the target gene refers to a sequence complementary to a sequence to be detected (base sequence) that specifies a gene to be detected, and a preferable length range is 7 to 40 bases. It is. In the gene detection probe of the present invention, the target gene recognition sequence is sandwiched between the repeating units of the complementary inverted repeat sequence. Because of such sequence characteristics, the gene detection probe according to the present invention has complementary reverse repeat sequences in the absence of the target gene or when the conditions for hybridization with the target gene are not sufficient. Forms a closed molecular structure composed of a double-stranded stem structure formed by hybridizing and a single-stranded loop structure composed of a recognition sequence of the target gene.
Therefore, “including a reverse repeat sequence complementary to each other in the molecule, and a sequence capable of recognizing a target gene between the repeat sequences” means that “from a double-stranded stem and a single-stranded loop alone. It can be said to be a typical example of a “sequence feature that takes a closed structure, but becomes an open structure by forming a double strand with the target gene under conditions where the target gene is present”.
The sequence capable of recognizing the target gene is preferably located in a single-stranded loop structure, and a part or all of one or both of the above repetitive sequences (parts constituting the stem) of the sequence capable of recognizing the target gene It may be included in some.
In addition, the sequence to be detected that specifies the target gene is a sequence that is included in a molecule that is actually used in a hybridization reaction with a probe. This target sequence may be the target gene itself to be detected, or may be a complementary sequence of a portion specific to the target gene contained in the target gene. Such a partial sequence may be a fragment obtained directly from the isolated gene, or may be synthesized based on a partial sequence selected from the base sequence of the target gene.
Such a structure consisting of a double-stranded stem structure and a single-stranded loop is generally called a hairpin structure. However, when the probe molecule is DNA or RNA, the stability of the structure is complementary to the intramolecular double-stranded formation. In addition to the type and length of the repetitive sequence, it is influenced by the temperature and salt concentration of the buffer in which the probe is present. For example, in the case of an oligonucleotide molecule consisting of 27 bases, 5m-TTGAG-3 '/ 5'-CTCAA-3' complementary reverse repeat sequence of 5 bases, Tm value related to intramolecular duplex formation Is estimated to be about 54 ° C. in a buffer containing 50 mM NaCl based on the actual measurement. The Tm value in duplex formation between the probe molecule and the target gene is determined by the type and length of the recognition sequence of the target gene. When the sequence is 20 mer or less, the Tm value is approximated by the following equation according to Wallace's law: Given. Tm (° C.) = 2 × (number of A or T) + 4 × (number of G or C). In the case of a sequence larger than 20 mer, several calculation methods such as Nearest-neighbor method are applied.
When the presence of the target gene is detected by the gene detection probe of the present invention, the Tm value in the double strand formation between the probe molecule and the target gene is equal to or higher than the Tm value in the double strand formation in the probe molecule. It is preferable to set. The preferred length of the target gene recognition sequence is 15 to 18 mer, but shorter or longer ones can be used depending on the purpose.
In the target gene analysis method of the present invention, the hairpin structure is maintained by the probe alone, and the reaction between the sample and the probe is performed under the condition that the hairpin structure is eliminated when the molecule having the sequence to be detected and the probe are hybridized. .
The salt concentration of the buffer solution affects the hybridization when the probe molecule is DNA or RNA. Since the double strand is stabilized by the presence of the cation, the Tm value generally increases at high ionic strength. On the other hand, when the probe molecule is composed of peptide nucleic acid, the influence of ionic strength is reduced.
When designing a recognition sequence for a target gene in the gene detection probe of the present invention, sequence information such as the base sequence of the target gene itself or the consensus sequence of the gene family to which the target gene belongs is necessary. The operation of selecting a recognition sequence having an appropriate Tm value from the sequence information can be performed by visual observation, but a commercially available primer design program or an online program provided by a public institution such as Oligo Calculator (http) : /Mbcf.dfci.harvard.edu/docs/oligocalc.html) and the like.
On the other hand, the complementary inverted repeat sequence can be arbitrarily set regardless of the target gene. In general, it is a sequence that contributes to the formation of a stable hairpin structure, a sequence that is not complementary to the recognition sequence of the target gene, and the Tm value in intramolecular duplex formation is It is preferable that the sequence gives a value equal to or lower than the Tm value in the formation of this strand, but depending on the purpose, a part or all of one or both of the repetitive sequences may be a recognition sequence of the target gene. May be included in some.
Many of the various primer design programs can calculate the ease of formation of intramolecular duplexes, and can therefore be used as a guide for sequencing.
In the target gene detection probe of the present invention, the redox unit is bound to one strand constituting the double-stranded stem in a state where the double-stranded stem is formed. When the two strands constituting the double-stranded stem have an extended portion from the double-stranded stem on the opposite side of the single-stranded loop of the double-stranded stem, one of the extended portions has a redox unit. May be combined. For example, the redox unit is preferably included in any one of the following positions a) and b).
a) On formation of a closed structure consisting of a double-stranded stem and a single-stranded loop, on any one of the strands constituting the double-stranded stem.
b) Either end of a linear nucleic acid or similar molecule.
The gene detection probe of the present invention alone can take a closed structure consisting of a double-stranded stem and a single-stranded loop, but under the condition that the detected sequence for specifying the target gene exists, It is characterized by having an open structure by forming a double strand. The region that forms the double-stranded stem has the largest structural change before and after the recognition of the target gene. In the present invention, such a structural change is used as a change in an electrical signal. Therefore, the redox unit is preferably contained on one strand forming a double-stranded stem in the gene detection probe of the present invention. As such a method, reverse repeat sequences complementary to each other are used. It is preferable to arrange on one of the repeating units. In the probe for gene detection of the present invention, it is preferable to arrange inverted repeat sequences complementary to each other at both ends of the probe, so that either end of a linear nucleic acid constituting the probe or a molecule similar thereto It is more preferable to contain a redox unit. Here, the “terminal” does not need to be strictly a terminal, and a width of 3 to 5 nucleotides is allowed. That is, the case where the redox unit is bound to the fifth nucleotide from the end of the molecule is also considered to be included in the “terminal”. In addition, when a linker molecule or the like is bound to the end of a linear nucleic acid or a similar molecule depending on the purpose, a redox unit may be included in the linker.
A preferred form in which the redox unit is contained in the probe molecule is a case where it is covalently bound to a linear nucleic acid or a similar molecule (nucleic acid analogue) directly or via a linker molecule. Although it is mentioned, it is not limited to this.
The redox unit bound to the gene detection probe of the present invention is a substance having a redox potential of +1.0 to -1.0 V in a single measurement (measurement in a state where it is not bound to a probe or the like). It is preferable. More preferably, selected from quinone compounds such as quinone and derivatives thereof, flavin compounds such as flavin and derivatives thereof, porphyrin compounds such as porphyrin and derivatives thereof, organometallic complexes represented by metallocene compounds such as ferrocene, metals and metal compounds. Can be used. Examples of the quinone compound include anthraquinone and pyrroloquinoline quinone. If necessary, a plurality of redox units may be bound to one probe.
FIG. 1 shows a schematic structure and a general chemical formula of a typical gene detection probe of the present invention. In the chemical formula, N represents an arbitrary nucleotide, A represents an adenosine nucleotide, and U represents a uridine nucleotide. Redox represents a redox unit. In the figure, 1 indicates the probe molecule body, and 2 and 8 indicate complementary inverted repeat sequences. Reference numeral 3 denotes a target gene recognition sequence, which forms a hairpin structure and forms a single-stranded loop. While 10 is a molecule composed of an oligonucleotide, 9 represents a linker composed of 6-mercaptohexanol. The redox unit shown in 4 indicates that it is covalently bound to the uracil base of the uridine nucleotide.
When the probe molecule (portion not including the redox unit of the gene detection probe of the present invention) is composed of DNA or RNA, it can be synthesized by a commercially available DNA / RNA synthesizer. In many cases, nucleic acids containing various modified nucleotides similar to DNA or RNA nucleotides can be synthesized using a commercially available DNA / RNA synthesizer.
When a peptide nucleic acid is contained in the probe molecule, it can be synthesized by a liquid phase method or a solid phase method in the same manner as a peptide, and the synthesized one is also commercially available. Regarding the method for synthesizing peptide nucleic acids, the specification of JP-T-6-509063, the specification of US Pat. E. Nielsen et al. , Journal of American Chemical Society, 114, 1895-1987 (1992), p. E. Nielsen et al. , Journal of American Chemical Society, 114, 9679-9678 (1992).
FIG. 2 illustrates a typical electrochemical gene detection method of the present invention. The most preferable form of gene detection in the present invention is performed in a state where the gene detection probe of the present invention is fixed to the electrode surface, and more preferably, a plurality of types of gene detection probes are arranged on a carrier or a substrate. This is performed in the state of a DNA chip fixed to the electrode.
In FIG. 2, the probe molecule is bonded to the surface of 5 electrodes via 6 thiol groups located at the end of a linker consisting of 9 6-mercaptohexanol. Recognizing the target gene 7 opens the hairpin structure and a double strand consisting of 7 and 1 is formed. Along with such a structural change, the four redox units change the distance from the electrode surface, and the change in the distance is measured as a change in the current value.
The “target nucleic acid detection chip” of the present invention is a single type or a plurality of types of gene detection probes of the present invention immobilized on an appropriate carrier, for example, the surface of an electrode placed on the surface of a substrate or substrate of various shapes. When using multiple types of probes, these may be fixed to a single electrode, or arranged in an orderly manner so that the positional information of each probe can be traced. The method of fixing to can be used.
When multiple different probes are used, the detected sequence hybridizes to which probe based on the obtained electrical responsiveness by changing the electrical responsiveness of the redox unit for each of multiple different probes that recognize different target genes. The target gene can be identified based on the information on the target gene recognition sequence of the probe hybridized with the sequence to be detected. For example, in order to obtain a system capable of detecting the first and second target genes, the first probe having a sequence complementary to a nucleic acid molecule having a first detected sequence capable of specifying the first target gene A second probe having a sequence complementary to a nucleic acid molecule having a second detected sequence capable of specifying a second target gene and having a redox unit having a different electrical response from the first probe, A region is fixed to a single electrode or two independent electrodes, and reacted with a sample. When the nucleic acid molecule having the first detected sequence is present in the sample, the first target gene can be detected by the change in the electrical response of the redox unit of the first probe, and the second target is detected in the sample. When the nucleic acid molecule having the detected sequence is present, the second target gene can be detected by the change in the electrical response of the redox unit of the second probe. When both of these nucleic acid molecules having the first and second detected sequences are present in the sample, the electrical response of the redox unit of the first probe changes, and the second probe different from this has Based on the change in the electrical response of the redox unit, the first and second target genes can be detected.
In the DNA chip of the present invention, it is preferable that the gene detection probe of the present invention is fixed to the electrode surface by bonding between any one of the following parts c) and d) and the electrode surface.
c) When forming a closed structure composed of a double-stranded stem part and a single-stranded loop part, one of the two chains constituting the double-stranded stem that does not contain a redox unit.
d) Terminals that do not contain redox units of linear nucleic acids or similar molecules.
The gene detection probe of the present invention alone has a closed structure consisting of a double-stranded stem portion and a single-stranded loop portion, but the target gene and the double strand under the condition that a target sequence for identifying the target gene exists. It is characterized by having an open structure by forming a chain. The DNA chip of the present invention is characterized in that gene detection is performed by reading a change in the distance of the redox unit as an electric current value before and after the recognition of the target gene. Therefore, it is preferable to fix the probe so that the change of the distance becomes the largest. For this purpose, it is preferable that one of the two strands constituting the double-stranded stem does not contain a redox unit. It is preferable to fix the terminal of the linear nucleic acid constituting the probe or a molecule similar thereto to the electrode without the redox unit. Here, the term “terminal” does not need to be strictly a terminal, and a width of 3 to 5 nucleotide units is allowed. In addition, when a linker molecule or the like is bonded to the end of a linear nucleic acid or a similar molecule depending on the purpose, the linker can be fixed by bonding the linker to the electrode surface. In the case of the probe shown in FIG. 1, immobilization is performed by binding the SH group of 6-mercaptohexanol bound to the 3 ′ end of the nucleotide chain to the electrode surface.
As shown in FIG. 3, the method of using a plurality of types of probes immobilized on a single electrode is a method that should be a feature of the present invention, in which the electrical response of each of a plurality of probes having different target genes is measured. This can be achieved by using different redox units.
For example, when a redox unit consisting of anthraquinone and ferrocene is used for probe 1 and probe 2 having different target genes, the peak of the current value in the hairpin state in differential pulse voltammography is It can be seen near -0.5V and with probe 2 around + 0.3V. Therefore, even if these probes are fixed on the same electrode, the target gene 1 or the target gene 2 can be distinguished from each other by reading the peak current value corresponding to each probe without changing the measurement conditions individually. Can be detected.
Such a single electrode type / multiple analysis DNA chip can be simplified in structure of the chip, and is expected to reduce the cost of chip production and measurement.
Materials for electrodes used in the electrochemical gene detection method of the present invention include carbon electrodes such as graphite and glassy carbon, noble metal electrodes such as platinum, gold, palladium and rhodium, titanium oxide, tin oxide, manganese oxide, and oxidation. Examples thereof include oxide electrodes such as lead, semiconductor electrodes such as Si, Ge, ZnO, and CdS, and electronic conductors such as titanium, but it is particularly preferable to use gold or glassy carbon. These electronic conductors may be coated with a conductive polymer or a monomolecular film.
As the carrier on which the electrode is placed, it is preferable to use a hydrophobic or low hydrophilic electrically insulating material. Examples of such a material include glass, cement, ceramics such as ceramics or new ceramics, polyethylene terephthalate, and cellulose acetate. , Polymers such as polycarbonate of bisphenol A, polystyrene, polymethylmethacrylate, silicon, activated carbon, porous glass, porous ceramics, porous silicon, porous activated carbon, woven / knitted fabric, nonwoven fabric, filter paper, short fiber, membrane filter, etc. Among them, various polymers, glass or silicon are particularly preferable. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the electrically insulating carrier or substrate is not particularly limited.
When a plurality of electrodes are arranged on the carrier or the substrate, it is preferable that each electrode is arranged on the carrier or the substrate regularly so as not to contact each other. Before providing the electrode, a layer made of a hydrophilic polymer substance having a charge or a layer made of a crosslinking agent may be provided on the carrier or the substrate. By providing such a layer, unevenness of the carrier or the substrate can be reduced. Further, depending on the type of the carrier or the substrate, it is possible to include a hydrophilic polymer substance having a charge in the substance, and a carrier or substrate subjected to such treatment can also be preferably used.
Various methods can be used as the method for immobilizing the probe to the electrode surface, but a method via a covalent bond is preferred. As such an immobilization method, there is a method in which the end of the probe is modified with a compound containing a thiol group, for example, 6-mercaptohexanol, and immobilized on the surface of the activated gold electrode by a thiol bond. In such binding, a spacer molecule of an appropriate size may be interposed between the probe molecule and the active group on the electrode surface.
The probe molecules are preferably immobilized by spotting an aqueous liquid on which the probe molecules are dissolved or dispersed on the electrode surface. The aqueous liquid containing the probe molecule may contain an additive that increases the viscosity of the aqueous liquid. Examples of such additives include sucrose, polyethylene glycol, glycerol and the like. After spotting, the probe molecules are fixed when left for several hours at a predetermined temperature. Although spotting can be performed by manual operation, it can also be performed using a spotter provided in a widely used DNA chip manufacturing apparatus. Incubation may be performed after spotting. After incubation, it is preferable to remove unfixed probe molecules by washing.
The DNA chip produced as described above can be used immediately for the nucleic acid to be examined.
In the present invention, various genes can be detected by changing the recognition sequence of the target gene in the gene detection probe. In the food field, when a gene detection probe that uses a recognition sequence that is complementary to the base sequence of all or part of a microorganism contained in food, direct detection of the microorganism contained in the food is performed. Can be performed, and food hygiene inspection becomes possible. Examples of microorganisms contained in such foods include pathogenic E. coli, staphylococci, and Salmonella.
In the field of agriculture, forestry and fisheries, it can be used for detection of pathogenic microorganisms or viruses in crops, livestock animals and fish, and verification of production areas and brands of agricultural products and livestock products. It can also be used to detect genetically modified crops and livestock products. When a probe whose sequence is complementary to a part of the base sequence of a plant virus or viroid is used, it can detect a plant virus or viroid infected with a plant, and diagnoses infectious diseases in the agricultural field. Is possible. Examples of such plant viruses or viroids include tobacco mosaic virus and cauliflower mosaic virus. Detection of pathogenic microorganisms or viruses that infect fish when using a probe whose recognition sequence is complementary to the base sequence of all or part of the pathogenic microorganisms or viruses that infect livestock animals and fish Infectious diseases can be diagnosed in the livestock and fisheries fields. Examples of such pathogenic viruses and microorganisms include foot-and-mouth disease virus and pathogenic Vibrio.
In the medical field, it is possible to detect pathogenic microorganisms or viruses that infect the human body and cause infections, genes causing genetic diseases, activated proto-oncogenes, and the like. Infectious disease diagnosis is possible when using a probe that recognizes a complementary sequence to the base sequence of all or part of a pathogenic microorganism or virus that infects the human body and causes infections, etc. as a nucleic acid probe become. Examples of such pathogenic microorganisms that infect human bodies and cause infections include Streptococcus, Mycoplasma, Clostridium, Chlamydia, Salmonella, herpes simplex, and cytomegalovirus, which are pathogenic microorganisms.
When a probe having a recognition sequence that is complementary to the entire base sequence of a gene causing a genetic disease or a part thereof is used, a direct test for a genetic disease is possible. Examples of the causative gene for such genetic diseases include adenosine deaminase deficiency and sickle cell anemia causative gene.
When a probe having a recognition sequence that is a sequence complementary to the whole or a part of the base sequence of the activated proto-oncogene is used, cancer diagnosis becomes possible. Examples of such activated proto-oncogenes include oncogenes described in the oncogene data book (Masashi Shibuya, Shujunsha).
The causative gene of a genetic disease, the susceptibility to other diseases, the ease of use of drugs, and the like may be determined by a single nucleotide polymorphism (SNP) on the gene. In the detection of such a gene, it is necessary not to detect the presence of the gene itself or the expression level of the gene, but to identify slight structural differences on the gene that are different for each specimen. In the present invention, it is possible to detect the presence or absence of SNP in a sample by using more stringent hybridization conditions.
In the gene detection method of the present invention, first, a nucleic acid to be examined is prepared by an extraction operation from various materials and biological tissues. When preparing DNA from living tissue, various DNA kits provided by reagent manufacturers, such as Get pure DNA Kit-Blood (manufactured by Dojin Chemical Co., Ltd.) suitable for preparation from blood, and ISOHAIR suitable for preparation from hair (Nippon Gene Co., Ltd.) can be used. The prepared nucleic acid can be amplified as necessary by, for example, polymerase chain reaction (PCR) or the like, or in the case of RNA, converted to the corresponding DNA by reverse transcription reaction.
Next, hybridization of the nucleic acid obtained by the above operation with the probe for gene detection of the present invention is performed using a DNA chip. In the present invention, chemical treatment such as labeling of nucleic acid is not required before hybridization, but when the nucleic acid sample is double-stranded DNA, it is denatured by heat treatment and used as a single strand for hybridization. It is preferable. Hybridization is carried out by bringing a sample solution in which nucleic acid is dissolved or dispersed into contact with the gene detection probe of the present invention. Hybridization is preferably carried out in the temperature range of 20 to 40 ° C. and in the range of 1 to 24 hours. However, depending on the chain length of the probe molecule immobilized on the electrode, the type of sample nucleic acid, etc. It is desirable to set optimal conditions. After completion of hybridization, washing is preferably performed to remove unreacted sample nucleic acid fragments.
In the gene detection method using the DNA chip of the present invention, the current value can be measured immediately after the hybridization with the nucleic acid sample without any special operation. The current amount can be measured by any method as long as the amount of current flowing between the electrode and the redox unit can be measured. Cyclic voltammography (CV), differential pulse voltammography (DPV), linear sweep voltammography, potentiostat, etc. are preferably used. It is particularly preferable to measure differential pulse voltammography by immersing the electrode on which the counter electrode and the gene detection probe are fixed in an electrolyte solution to form a pair of electrolytic systems. It is particularly preferred to measure a differential pulse voltammography.
In DPV, a peak current at a potential unique to the probe is obtained according to the redox potential of the redox unit used for the probe. This peak current is maximum when the probe has a hairpin structure, and is minimum when the probe and the target gene form a double strand. In the case of a DNA chip using a redox unit with different electrical responses for each target gene and a plurality of gene detection probes fixed to a single electrode, a unique potential that gives a peak current for each probe Therefore, it is possible to detect each target gene by tracing the change of the peak current at these potentials in the DPV.
In the reaction between the sample and the probe, the hairpin structure of the unreacted probe is maintained, the solvent of the reaction system that forms a double strand with the target sequence, and the current in the double strand formation between the probe and the target sequence. As a solvent in the detection based on 1-100 mM MgCl ₂ It is desirable to use a neutral to weakly basic buffer solution containing Using a buffer solution (pH 8.0) consisting of 10 mM Tris-HCl and 1 mM EDTA, MgCl ₂ It is particularly desirable to use a concentration in the range of 3-10 mM.

ＯＤＮ２〜ＯＤＮ４は配列表の配列番号２〜４に示す配列からなり、それぞれＤＮＡ合成機により合成した。

（３）プローブで修飾された金電極の作成
表面積２ｍｍの金電極を２Ｍ水酸化カリウム溶液中で３時間煮沸し、純水で洗浄した後、濃硝酸溶液に室温で３時間浸し、さらに純水で洗浄した。上記の処理をした金電極の表面に１μＭのＯＤＮ１溶液を１μＬ滴下し、ゴムキャップをかぶせて室温で３時間放置した。その後、電極を軽く純水で洗浄し、１ｍＭのメルカプトヘキサノール１μＬを滴下し、ゴムキャップをかぶせて室温で３時間放置した。
（４）ハイブリダイゼーション
ＯＤＮ１を固定化した金電極上に５０ナノモル相当のＯＤＮ２〜４をそれぞれ含む、５ｍＭリン酸ナトリウム、５０ｍＭ塩化ナトリウム、ｐＨ７．０の溶液を滴下し、室温で１時間放置することによりハイブリダイゼーションさせた。その後、緩衝溶液で軽く洗浄を行った。
（５）電流値の測定
ディファレンシャルパルスボルタンメトリー（ＤＰＶ）による測定は、ＡＬＳ社製のモデル６６０Ａ エレクトロケミカルアナライザーで行った。参照電極は飽和カロメア電極（ＳＣＥ）、対極はプラチナ電極をそれぞれ用いた。測定は、５ｍＭリン酸ナトリウム、５０ｍＭ塩化ナトリウム、ｐＨ７．０の溶液中、室温で行い、ｐｕｌｓｅ ｐｅｒｉｏｄを２００ｍｓ、ｓｃａｎ ｒａｔｅを１００ｍＶ／ｓ、ｐｕｌｓｅ ａｍｐｌｉｔｕｄｅを５０ｍＶ、ｐｕｌｓｅ ｗｉｄｔｈを５０ｍｓとした。それぞれの検体用オリゴヌクレオチドについて、２回づつの測定を行い、値を平均化した。
プローブＯＤＮ１における認識配列と完全にマッチする配列を含むＯＤＮ２を検体として用いた場合のＤＰＶ結果を図５に示す。検体を加えない状態（ヘアピン）で認められたピーク電流値が、検体を加えた状態（ＷＴ）では顕著に減少していることがわかる。
一方、プローブＯＤＮ１における認識配列とマッチしない配列からなるＯＤＮ３、ＯＤＮ４の場のＤＰＶ結果を夫々、図６、７に示す。いずれの場合も、検体を加えない状態（ヘアピン）及び検体を加えた状態（Ｇ１７８ＭＵまたはＦ５０８）共に電流応答が見られるが、検体を加えた状態において、電流量、ピーク値共に減少している。これは、検体の添加によって、プローブのヘアピン構造が多少の影響を受けていることを示唆するものと思われるが、ハイブリダイゼーションの条件を調整することにより、電流値の変化は抑えられるものと思われる。
図８、９には各検体ごとに、添加の前後における電流量、ピーク電流の変化を相対的に示した。これらの図からもわかるように、プローブにおける認識配列と完全にマッチする場合は、その認識の前後において顕著な電流応答の変化が認められる。マッチしない配列からなる検体の場合は、その認識の前後において電流値の変化がほとんどないことが望ましいが、本実施例におけるハイブリダイゼーションの条件では、プローブと検体との間での相互作用を排除することができなかったため、電流値の差異を生じたものと思われる。しかしながら、図に示される電流値の差異があれば検出には支障はない。(1) Synthesis of anthraquinone-modified uridine nucleotides A synthetic route is shown in FIG.
Acid chloride (compound 2) was obtained from 2-anthraquinonecarboxylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) by thionyl chloride treatment. Compound 2 and propargylamine (1, Wako Pure Chemical Industries, Ltd.) (weight ratio 1: 1) in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1 equivalent, Wako Pure Chemical Industries, Ltd.) The mixture was reacted in N, N-dimethylformamide by stirring at room temperature for 4 hours, extracted, and purified by column chromatography to obtain the product 3 (21%).
5-Iodo-2′-deoxyuridine (4, manufactured by Sigma) and 4,4′-dimethoxytrityl chloride (manufactured by Tokyo Chemical Industry) (weight ratio of 1: 1) were mixed in pyridine and stirred at room temperature for 16 hours. After reaction, extraction and purification by column chromatography gave the product 5 (65%).
Products 3 and 5 (weight ratio 1 to 1) were added in the presence of (tetrakistriphenylphosphine) palladium (0.15 equivalent, manufactured by Wako Pure Chemical Industries) and triethylamine (1 equivalent, manufactured by Wako Pure Chemical Industries, Ltd.). Reaction was carried out by stirring for 12 hours at room temperature in dimethylformamide, followed by extraction and purification by column chromatography to obtain the product 6 (74%). Further, the product 6 and 2-cyanoethyltetraisopropyl phosphorodiamidite (Aldrich) (weight ratio 1: 1) were stirred in acetonitrile at room temperature for 1 hour in the presence of tetrazole (1 equivalent, manufactured by Dojindo). The product 8 obtained was used for DNA synthesis as it was.
(2) Preparation of Probe and Sample Oligonucleotide Probe ODN1 has a nucleotide sequence (TGAGTATCATCTTTGGGTTTTCTCAA) shown in SEQ ID NO: 1 in the sequence listing. This oligonucleotide was synthesized with a DNA synthesizer, and the anthraquinone modified uridine prepared in (1) was added to the 5 ′ end. After purification of the synthesized product, 6-mercaptohexanol was added to the 3 ′ end.

ODN2 to ODN4 consisted of sequences shown in SEQ ID Nos. 2 to 4 in the sequence listing, and were synthesized by a DNA synthesizer.

(3) Preparation of a gold electrode modified with a probe A gold electrode having a surface area of 2 mm was boiled in 2M potassium hydroxide solution for 3 hours, washed with pure water, then immersed in concentrated nitric acid solution at room temperature for 3 hours, and further purified water Washed with. 1 μL of a 1 μM ODN1 solution was dropped on the surface of the gold electrode subjected to the above treatment, and a rubber cap was put on the surface to stand at room temperature for 3 hours. Thereafter, the electrode was lightly washed with pure water, 1 μL of 1 mM mercaptohexanol was dropped, and the rubber cap was put on and left at room temperature for 3 hours.
(4) Hybridization A solution of 5 mM sodium phosphate, 50 mM sodium chloride, pH 7.0, each containing 50 nmoles of ODN2-4, is dropped on a gold electrode on which ODN1 is immobilized, and left at room temperature for 1 hour. Hybridization. Thereafter, it was gently washed with a buffer solution.
(5) Measurement of current value Measurement by differential pulse voltammetry (DPV) was performed with a model 660A electrochemical analyzer manufactured by ALS. The reference electrode was a saturated calomere electrode (SCE), and the counter electrode was a platinum electrode. The measurement was performed at room temperature in a solution of 5 mM sodium phosphate, 50 mM sodium chloride, pH 7.0, the pulse period was 200 ms, the scan rate was 100 mV / s, the pulse amplitude was 50 mV, and the pulse width was 50 ms. For each specimen oligonucleotide, two measurements were taken and the values were averaged.
FIG. 5 shows the DPV result when ODN2 containing a sequence that perfectly matches the recognition sequence in probe ODN1 is used as a specimen. It can be seen that the peak current value observed when no specimen is added (hairpin) is significantly reduced when the specimen is added (WT).
On the other hand, FIGS. 6 and 7 show the DPV results in the fields of ODN3 and ODN4, which are sequences that do not match the recognition sequence in the probe ODN1, respectively. In either case, a current response is observed in both the state where the sample is not added (hairpin) and the state where the sample is added (G178MU or F508), but both the amount of current and the peak value are decreased in the state where the sample is added. This seems to suggest that the hairpin structure of the probe is somewhat affected by the addition of the sample, but it seems that the change in the current value can be suppressed by adjusting the hybridization conditions. It is.
8 and 9 relatively show changes in the amount of current and the peak current before and after the addition for each specimen. As can be seen from these figures, a significant change in the current response is observed before and after the recognition when it completely matches the recognition sequence in the probe. In the case of a specimen consisting of a sequence that does not match, it is desirable that there is almost no change in the current value before and after the recognition, but the hybridization conditions in this example exclude the interaction between the probe and the specimen. It was thought that the difference of the current value was caused because it was not possible. However, if there is a difference between the current values shown in the figure, there is no problem in detection.

フラビン類縁体は、Ｉｋｅｄａら、Ｔｅｔｒａｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ ４２、２５２９（２００１）に示した方法で合成した。
自動ＤＮＡ合成機により、３’端（サポートのヌクレオシド）より２番目にＴｈｉｏｌ ｍｏｄｉｆｉｅｒ Ｃ６ Ｓ−Ｓ（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ製）を導入し、５’端にＡｍｉｎｏ ｍｏｄｉｆｉｅｒ Ｃ２ ｄＴ（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ製）を導入する方法で配列番号５の配列（ＴＡＧＣＧＡＡＡＴＡＡＧＴＡＴＴＡＧＡＣＡＡＣＣＧＣＴＡ）を有するオリゴＤＮＡを合成した。サポートからの切り出し、脱保護の後、２０ｍＭのＮ−ヒドロキシスクシンイミドで活性化されたフラビン類縁体を含む５０ｍＭリン酸ナトリウム緩衝液（ｐＨ８．７）で、１２時間攪拌した。反応液をＨＰＬＣにより精製することで、５’端にフラビン類縁体が付加したオリゴＤＮＡを得た。さらに、これを０．１ＭのＤＴＴ溶液中（５０ｍＭ Ｎａ ｐｈｏｓｐｈａｔｅ，ｐＨ８．４）で３０分間撹拌してＳ−Ｓ結合の還元を行い、ＨＰＬＣ精製することで、３’端をヘキサンチオール化したオリゴＤＮＡ（ＯＤＮ５）を得た（図１０）。
（２）プローブで修飾された金電極の作成、ハイブリダイゼーション
実施例１に記載の方法で行った。
（３）ＤＰＶによる電流値の測定は実施例１に記載の方法で行った。
プローブＯＤＮ５における認識配列と相補的なＯＤＮ３（Ｇ１７８ＭＵ）を検体として用いた場合のＤＰＶ結果を図１２に示す。検体を加えない状態（ヘアピン）で認められたピーク電流値が、検体を加えた状態（Ｇ１７８ＭＵ）では顕著に減少していることがわかる。
一方、プローブＯＤＮ５における認識配列と相補的でないＯＤＮ２（ＷＴ）、ＯＤＮ４（Ｆ５０８）を検体とした場合のＤＰＶ結果を夫々、図１１、１３に示す。いずれの場合も、検体を加えない状態（ヘアピン）及と検体を加えた状態（ＷＴまたはＦ５０８）で電流応答がほとんど変化していない。(1) Preparation of flavin analog-modified probe ODN5 containing a flavin analog was synthesized as a redox unit.

Flavin analogs were synthesized by the method shown in Ikeda et al., Tetrahedron Letters 42, 2529 (2001).
Method of introducing Thiol modifier C6 SS (manufactured by Glen Research) second from the 3 ′ end (support nucleoside), and introducing amino modifier C2 dT (manufactured by Glen Research) to the 5 ′ end by an automatic DNA synthesizer Thus, an oligo DNA having the sequence of SEQ ID NO: 5 (TAGCGAAATAAGTATAGATACAACCGCTA) was synthesized. After cutting out from the support and deprotecting, the mixture was stirred for 12 hours with 50 mM sodium phosphate buffer (pH 8.7) containing a flavin analog activated with 20 mM N-hydroxysuccinimide. The reaction solution was purified by HPLC to obtain oligo DNA with a flavin analog added to the 5 ′ end. Furthermore, this was stirred for 30 minutes in a 0.1 M DTT solution (50 mM Na phosphate, pH 8.4) to reduce the S—S bond, and purified by HPLC to obtain an oligo-thiolated 3 ′ end oligo. DNA (ODN5) was obtained (FIG. 10).
(2) Preparation and hybridization of a gold electrode modified with a probe The method described in Example 1 was used.
(3) The measurement of the current value by DPV was performed by the method described in Example 1.
FIG. 12 shows the DPV result when ODN3 (G178MU) complementary to the recognition sequence in the probe ODN5 is used as a sample. It can be seen that the peak current value observed when no sample is added (hairpin) is significantly reduced when the sample is added (G178MU).
On the other hand, FIGS. 11 and 13 show the DPV results when ODN2 (WT) and ODN4 (F508) that are not complementary to the recognition sequence in the probe ODN5 are used as samples, respectively. In either case, the current response hardly changes between the state in which the specimen is not added (hairpin) and the state in which the specimen is added (WT or F508).

プローブの配列が異なるため一概には比較できないが、アントラキノン修飾型プローブに比較し、フラビン修飾型プローブの場合は、相補的な配列に対してＳ／Ｎ比が高く、相補的でない配列に対してＳ／Ｎ比が低い理想的な結果となった。The S / N ratio in gene detection was calculated and compared for the anthraquinone modified probe used in Example 1 and the flavin modified probe used in Example 2. Here, defined as a value obtained by dividing the S / N of the current before hybridization ratio _{(Ap before)} the amount of current after hybridization from _{(Ap afeter)} value of current before hybridization minus _{(Ap before)} did. The calculation results are shown in Table 1 and FIG.

Since the probe sequences are different, it is not possible to compare them in general. However, compared to anthraquinone modified probes, the flavin modified probes have a higher S / N ratio for complementary sequences and non-complementary sequences. An ideal result was obtained with a low S / N ratio.

Change in current value when DNA modified electrode was used repeatedly Hybridization / denaturation was repeated using the ODN5 modified electrode prepared in Example 2 to examine the change in current value. ODN3 (G178MU), ODN2 (WT), and ODN4 (F508), which are complementary targets, were added to the ODN5-modified electrode for hybridization, and the current value was measured by DPV. Subsequently, the electrode was immersed in a 90% formamide / 10% TE buffer solution for 1 minute to dissociate the double-stranded state, and after 1 minute in the TE buffer, the current value was measured by DPV. FIG. 15 shows the results of current values when the above operation was repeated for 10 cycles. When the complementary target ODN3 (G178MU) is used, current values reflecting the hairpin state and the double-stranded state are shown before and after hybridization, and these values are almost constant even after repeated cycles. there were. On the other hand, when ODN2 (WT) or ODN4 (F508) that is not complementary to the probe was used, although a slight decrease in the current value was observed in the second half of the cycle, the current value periodically reflecting hybridization / denaturation was observed. No significant change was seen. From the above results, it was shown that gene detection can be repeated with good reproducibility by using an electrode on which the gene detection probe of the present invention is fixed.

By using a hairpin probe having a redox unit according to the present invention and an electrochemical gene detection method using the same,
1) Detection of target nucleotide sequence and single nucleotide polymorphism,
2) Chip for detecting electrochemical target gene,
4) A biosensor or the like can be provided.

[Sequence Listing]

Claims (11)

A probe for detecting a target gene comprising a linear molecule comprising a recognition sequence capable of recognizing a target gene and comprising at least one of a nucleic acid and a nucleic acid analog, and a redox unit,
Independently, it comprises a double-stranded stem and a single-stranded loop, the single-stranded loop forms a hairpin structure closed by the double-stranded stem, and the detected sequence indicating the presence of the target gene and the recognition sequence Forming a double strand, the hairpin structure is opened, and a portion of the probe has a sequence necessary to form a double-stranded portion with a detected sequence, and the oxidation The reducing unit is a probe for detecting a target gene, wherein when the hairpin structure is formed, the reducing unit is bound to one of two strands that are bound or adjacent to each other except the single-stranded loop. The sequence capable of forming the hairpin structure has two inverted repeat sequences complementary to each other and a target gene recognition sequence consisting of a sequence complementary to the detected sequence provided between these repeat sequences. The probe for detecting a target gene according to claim 1. The probe for detecting a target gene according to claim 1 or 2, wherein at least a part of the single-stranded molecule is DNA or RNA. The probe for gene detection according to claim 1 or 2, wherein at least a part of the single-stranded molecule is a peptide nucleic acid. The probe for gene detection according to any one of claims 1 to 4, wherein the redox unit is contained at any one of the following positions a) and b).
a) One of the two strands constituting the double-stranded stem.
b) One end of the linear molecule. The probe for gene detection according to any one of claims 1 to 5, wherein the redox unit is a quinone compound, a metallocene compound, a flavin compound, a porphyrin compound, a metal or a metal compound. A target gene detection chip comprising a carrier having an electrode surface and the target gene detection probe according to any one of claims 1 to 6 bound to the electrode surface,
When the hairpin structure is formed, the end region of the strand that is not bound to the reducing unit of the two strands that are bound or adjacent to each other other than the single-stranded loop is bound to the electrode. A chip for detecting a target gene. Different probes capable of specifying each of a plurality of different target genes are immobilized on the electrode surface for each probe, and redox units having different electrical responses for each of the plurality of target genes are attached to each probe. The chip for detecting a target gene according to claim 7, which is bound. In a method for detecting a target gene using a probe,
Forming on the electrode surface of the target gene detection chip according to claim 7 a reaction system in which the probe fixed on the electrode surface can form the hairpin structure;
In the reaction system, when the sample and the probe contain a sequence to be detected, a double strand is formed by hybridization of the probe and the sequence to be detected. A step of reacting under the condition that the hairpin structure of can be opened and converted into a single strand;
A method of detecting a target gene, comprising: applying a potential to the electrode of the chip that has undergone the reaction, and determining a state of hybridization between the probe and the sequence to be detected based on the obtained current value . The method for detecting a target gene according to claim 9, wherein a plurality of probes that recognize a plurality of different target genes are immobilized on different electrodes for each probe. In a method for detecting a target gene using a probe,
Forming on the electrode surface of the target gene detection chip according to claim 8 a reaction system in which the probe fixed on the electrode surface can form the hairpin structure;
In the reaction system, when the sample and the probe contain at least one kind of detected sequences corresponding to a plurality of different target genes, the probe and the detected sequence are high. Forming a double strand by hybridization, and reacting under a condition in which the hairpin structure of the probe is opened and converted into a single strand having no double-stranded portion;
Applying the potential to the electrode of the chip that has undergone the reaction, and determining the state of hybridization between the probe and the sequence to be detected based on the obtained current value. Detection method.

Images