JP3917640B2 - Method for detecting presence of target nucleic acid using nucleic acid probe-immobilized substrate - Google Patents

Description

  The present invention relates to a nucleic acid probe-immobilized substrate for detecting the presence of a target nucleic acid and a nucleic acid detection method using the same.

  With the development of genetic engineering in recent years, it has become possible to diagnose and prevent diseases caused by genes in the medical field. These are called genetic diagnosis. For example, by detecting a defect or change in a human gene that causes a disease, it is possible to diagnose or predict the disease before the onset of the disease or at an extremely early stage. Along with the decoding of the human genome, research on the relationship between genotypes and diseases is progressing, and treatment tailored to each individual's genotype (tailor-made medicine) is becoming a reality. Therefore, it is very important to easily detect genes and determine genotypes.

  What is attracting attention in such gene analysis is an apparatus generally referred to as a DNA chip or a DNA microarray (herein, both are collectively referred to as a DNA chip). A DNA chip is an apparatus having a large number of nucleic acid probes having various base sequences fixed on a substrate. By using such a DNA chip, it is possible to detect many types of target nucleic acids in a single test. However, while having such advantages, the efficiency of hybridization of different target nucleic acids present in the sample is different, and in some cases, a very low hybridization efficiency may be obtained.

In view of the above situation, an object of the present invention is to provide a method for detecting the presence of a target nucleic acid in a sample nucleic acid using a probe-immobilized substrate capable of highly efficient hybridization.

The above object can be achieved by the following aspects of the present invention. That is,
(1) A nucleic acid probe-immobilized substrate comprising an electrode capable of performing electrochemical detection and having a nucleic acid probe immobilized on the electrode via a spacer, wherein the length of the spacer is X, The length from the substrate-side end of the hybridization site to the substrate-side end of the target nucleic acid when a target nucleic acid having a total length of 70 bases or more including a base sequence complementary to the nucleic acid probe is hybridized to the nucleic acid probe Is a method for detecting the presence of a target nucleic acid in a sample nucleic acid using a nucleic acid probe-immobilized substrate in which X ≧ Y, Y ≧ 10Å, and 200Å ≧ X are satisfied,
The sample nucleic acid is amplified using the selected primers so that the relationship between X and Y in the sample nucleic acid satisfies X ≧ Y, Y ≧ 10Å, and 200Å ≧ X, and the total length is 70 bases or more. Process,
Under conditions suitable hybridization can be obtained, and reacting the amplification product obtained by the amplification, the immobilized nucleic acid probe to the nucleic acid probe-immobilized substrate,
A method for detecting the presence of the target nucleic acid, comprising: electrochemically detecting the hybridization produced by the reacting step and determining that the target nucleic acid is present in the sample nucleic acid. And ( 2 ) a method for detecting the presence of a target nucleic acid having a total length of 70 bases or more including the target sequence, using the nucleic acid probe-immobilized substrate according to (1),
(A) When the target nucleic acid is hybridized with the nucleic acid probe with a target sequence, the target nucleic acid is positioned so that the base end of the target nucleic acid is located within 40 bases from the base end of the target sequence site. Preparing primers for amplifying nucleic acids;
(B) amplifying a sample nucleic acid using the primer prepared in (a) above;
(C) making the amplification product obtained in (b) single-stranded,
(D) reacting the single strand obtained in (c) with the nucleic acid probe;
(E) detecting the presence of the target nucleic acid in the sample nucleic acid by electrochemically detecting the hybridization produced in (d);
A method for detecting the presence of a target nucleic acid comprising:
It is.

Nucleic acid detection method using the nucleic acid probe-immobilized substrate of the present invention can provide a highly efficient hybridization.

1. SUMMARY OF THE INVENTION The present invention is basically a nucleic acid probe-immobilized substrate comprising a substrate and a nucleic acid probe fixed to the substrate via a spacer. The present invention is based on the finding that the present inventors can obtain more efficient hybridization by specifying the length of the spacer.

  That is, in the nucleic acid probe-immobilized substrate according to the embodiment of the present invention, when the length of the spacer is X and a target nucleic acid that includes the target sequence as a part of the nucleic acid probe is hybridized, the hybridization site The nucleic acid probe is fixed to the substrate via a spacer that satisfies the relationship X ≧ Y, where Y is the length from the substrate-side end of the target nucleic acid to the substrate-side end of the target nucleic acid.

  The measurement principle of the nucleic acid probe-immobilized substrate according to the embodiment of the present invention is as follows. The nucleic acid probe is designed to have a sequence complementary to the target sequence. When the target sequence is present in the sample nucleic acid, hybridization occurs on the nucleic acid probe-immobilized substrate. Therefore, the device of the present invention basically detects the presence of a double-stranded nucleic acid generated as a result of this hybridization, or detects the presence of a nucleic acid hybridized to the nucleic acid probe, It is possible to detect the presence of the target sequence in the sample nucleic acid. As used herein, the term “detecting hybridization” refers to detecting the resulting double-stranded nucleic acid or labeling a sample nucleic acid with any labeling substance in advance, and then labeling after hybridization. It is a general term for detecting a signal derived from a substance, or detecting the presence of a double-stranded nucleic acid or occurrence of hybridization by other means known per se.

  Such hybridization can be detected by, for example, electrochemical detection or fluorescence detection as described later.

  In the case of such electrochemical detection, the nucleic acid probe-immobilized substrate is basically immobilized via a desired nucleic acid probe and a spacer on an electrode arranged to detect an electrochemical signal on the substrate. Can be obtained.

  In addition, when a means for performing detection using a labeling substance such as a fluorescent substance is performed, a nucleic acid probe-immobilized substrate can be basically obtained by immobilizing a desired nucleic acid probe on a substrate via a spacer. It is done.

2. Explanation of Terms As used herein, the term “nucleic acid” refers to ribonucleic acid (ie RNA), deoxyribonucleic acid (ie DNA), peptide nucleic acid (ie PNA), methyl phosphonate nucleic acid, S-oligo, cDNA It is a general term for nucleic acids and nucleic acid analogues, such as and cRNA, and any oligonucleotides and polynucleotides. Moreover, such a nucleic acid may be naturally occurring or artificially synthesized.

  Here, the “nucleic acid probe” refers to a nucleic acid fragment containing a base sequence complementary to a target sequence and immobilized on a substrate. The nucleic acid probe has a sequence that is complementary to the target sequence of interest, and is thereby capable of hybridizing to the target sequence under appropriate conditions.

  Here, the “target sequence” refers to a base sequence whose presence is to be detected or a sequence to be captured by the base sequence of a nucleic acid probe. A nucleic acid containing such a target sequence is referred to as a target nucleic acid.

  The terms “complementary”, “complementary” and “complementarity” as used herein may be complementary in the range of 50% to 100%, preferably 100% complementary.

  As used herein, the term “spacer” refers to a chain substance having a certain length arranged between a nucleic acid probe and a substrate. When the substance constituting the spacer is a nucleic acid, a portion complementary or hybridized to the target sequence is classified as a probe, and the other portion is classified as a spacer.

As used herein, the term “spacer length” refers to the length of a chain molecule disposed between a nucleic acid probe and a substrate. When a blocking agent as shown in FIG. 3 is used for the substrate, the “spacer length” is obtained by subtracting the blocking agent length from the spacer length.

3. Aspects of the Invention First, an example of a basic configuration of the present invention will be described below.

(1) First Aspect The first aspect of the present invention will be described with reference to FIG. The nucleic acid probe-immobilized substrate 1 according to the first aspect of the present invention includes a nucleic acid probe 5 immobilized on a electrode 3 provided on a substrate 2 via a spacer 4 (FIGS. 1A and 1B). The electrode 3 is connected to a pad 6 for taking out electrical information. In FIG. 1B, for convenience, the spacer 4 is indicated by a thick line, and the nucleic acid probe 5 is indicated by a chain line.

  Such a nucleic acid probe-immobilized substrate 1 can be produced, for example, by placing an electrode on a silicon substrate by means known per se and immobilizing the nucleic acid probe on the electrode surface via a spacer. Is possible.

  In this embodiment, the number of electrodes is six, but the number of electrodes arranged on one substrate is not limited to this. Further, the arrangement pattern of the electrodes is not limited to that shown in FIG. 1A, and those skilled in the art can appropriately change the design as necessary. You may provide a reference electrode and a counter electrode as needed. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.

(2) Second Aspect FIG. 2 schematically shows a second aspect of the present invention. The nucleic acid probe-immobilized substrate 11 according to the second aspect of the present invention includes a nucleic acid probe 14 immobilized on a substrate 12 via a spacer 13 (FIG. 2). Also in FIG. 2, for convenience, the spacer 13 is indicated by a thick line, and the nucleic acid probe 14 is indicated by a chain line.

  Such a nucleic acid probe-immobilized substrate 11 can be produced, for example, by immobilizing a nucleic acid probe on a silicon substrate via a spacer by means known per se.

  In this embodiment, the number of nucleic acid probes arranged on one substrate is not limited to this, and may be changed as desired. Nucleic acid probes having a plurality of types of base sequences are arranged on one substrate. You may arrange. A person skilled in the art can appropriately change the design of the solid phase pattern on the substrate of plural and / or plural kinds of nucleic acid probes as necessary. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.

4). Configuration The embodiment according to the present invention has the basic configuration as described above, but is characterized in that the nucleic acid probe is fixed via a spacer. Specifically, the spacer used in the present invention has a spacer length X, and when a target nucleic acid containing the target sequence as a part of the nucleic acid probe is hybridized, It is a spacer that satisfies the relationship X ≧ Y, where Y is the length from the substrate-side end to the substrate-side end of the target nucleic acid.

  FIG. 3 shows an example of the binding state between the nucleic acid probe and the target nucleic acid. The left side of FIG. 3 schematically shows an example of the nucleic acid probe-immobilized substrate 30 and a general target nucleic acid. A linker agent 33a and a blocking agent 33b are treated on the surface of the electrode 32 disposed on the substrate 31. The nucleic acid probe 35 is fixed to the electrode 32 through the spacer 34 by the linker agent 33a. A target nucleic acid 36 having a target sequence complementary to the sequence of the nucleic acid probe 35 in a part thereof is shown next to it.

  A target nucleic acid containing a target sequence to be detected among a sample nucleic acid obtained from a sample obtained from a subject such as an individual, tissue or cell, or a sample obtained by processing it as desired. However, the position where the target nucleic acid exists is considered to be various. The sample nucleic acid 36 in the left diagram of FIG. 3 is shown here as an average example of such various target nucleic acids.

  The state when the nucleic acid probe 35 thus immobilized and the target nucleic acid 36 are hybridized is shown on the right side of FIG. As is clear from the figure, the length of the spacer 34 is “X”, and when the target nucleic acid 36 is hybridized to the nucleic acid probe 35 via the target sequence, the end of the target sequence portion from the substrate side end. The length to the end of the target nucleic acid on the substrate side is “Y”.

  When X <Y, the efficiency of hybridization between the nucleic acid probe 35 having a sequence complementary to the target sequence and the target nucleic acid 36 is low. That is, in such a case, considering the length of the target nucleic acid and the position of the target sequence in the target nucleic acid, the nucleic acid probe to be immobilized exists too close to the substrate surface. For this reason, the nucleic acid probes can cause steric hindrance to each other, and a solid substrate can cause steric hindrance. As a result, it is considered that sufficient hybridization efficiency between the nucleic acid probe and the target nucleic acid cannot be obtained.

  On the other hand, when X ≧ Y, the efficiency of hybridization between the nucleic acid probe 35 having a sequence complementary to the target sequence and the target nucleic acid 36 is high. In the case of X ≧ Y, as can be seen from the right diagram of FIG. 3, when the nucleic acid probe and the target sequence are hybridized, the surplus in the portion of the target nucleic acid 36 present on the substrate 31 side relative to the target sequence is temporarily If so, it is short or there is no such surplus. Further, since the spacer 34 has a sufficient length even when viewed from the length of the target nucleic acid and the position of the target sequence, the nucleic acid probe has a wide range of movement in the reaction solvent (that is, the degree of freedom is large). It is believed that the probability of encountering the target sequence during the hybridization reaction is improved.

  The relationship between X and Y is shown in FIG. In the graph of FIG. 5, the horizontal axis is the length of X, and the vertical axis is the length of Y. The center straight line is a graph of Y = X. A preferred relationship between Y and X according to embodiments of the present invention is X ≧ Y. In the figure, region A is a region that satisfies X ≧ Y in order to reduce the steric hindrance between the target nucleic acid and the solid phase. Region B is a region that is included in region A and further satisfies X−50Å ≧ Y in order to further improve the degree of freedom of the nucleic acid probe. The region C is a region that is included in the B region and is desirable in consideration of the cost and yield in the synthesis of the nucleic acid probe. The region D is a region that should be avoided even when Y is shorter than several tens of kilometers, considering that X needs a certain length in order to secure the degree of freedom. Further, the region E is a region in which Y is 0 or extremely short, so that the presence or absence of the spacer and the length do not affect the efficiency of hybridization.

  When the detection is actually performed, the regions A, B, C, and D are often used, but the regions C and D are more preferable in the embodiment of the present invention.

  In accordance with the spirit of the present invention, the limit of the length of X may be 20000 mm or less or 10,000 m or less. The upper limit of the length of X when considering from the aspect of synthesizing the nucleic acid probe may be 2000 Å (this corresponds to about 400 bases in nucleic acid), preferably 1000 、, more preferably 500 Å. That is, if the length of X is set long, the yield and purity in the case of synthesizing a nucleic acid probe may decrease.

  In addition, in order to satisfy the conditions according to the above-described aspects of the present invention, a device is selected when selecting a target sequence, or a primer sequence used when a target nucleic acid containing the target sequence is amplified from a sample is selected. It is also possible to devise when. In addition to adjusting the length of the spacer, it is possible to achieve better hybridization efficiency by making such adjustments.

  Examples of the substance used as the spacer may be organic chain molecules, such as nucleic acids, alkanes and polyethylene glycol, polypeptides, and the like.

  For example, when the spacer is a nucleic acid spacer composed of a nucleic acid, the base sequence is preferably a sequence that does not bind to a target nucleic acid or a nucleic acid that can be contained in a sample. Moreover, when detecting hybridization produced by electrochemical detection as described later, it is preferable to adopt a sequence that takes into account the binding tendency with the nucleobase of the double-stranded recognizer used therein. For example, Hoechst 33258 hardly binds to cytosine and guanine, and easily binds to thymine and adenine. On the other hand, a continuous sequence of guanine is difficult to synthesize. Therefore, a base sequence containing only cytosine or containing a lot of cytosine is more preferable, a base sequence consisting of or containing a lot of thymine is preferable, and a base sequence containing only guanine or adenine or containing a lot of them is less preferable.

  By arranging such a spacer, efficient hybridization between the nucleic acid probe and the target nucleic acid is achieved.

  The nucleic acid probe used in the present invention may have a length generally used as a probe. For example, the length of the nucleic acid probe may be about 3 bases to about 1000 bases long, and preferably about 10 bases to about 200 bases long.

  The substrate that can be used in the present invention may be any substrate on which a nucleic acid probe for hybridization with a target sequence is immobilized. Examples of such substrates can be, for example, non-porous, rigid and semi-rigid materials, even plates with wells, grooves or flat surfaces, and from three-dimensional shapes such as spheres and cubes. The form which becomes may be sufficient. The substrate may be made of, but not limited to, silica containing substrates such as silicon, glass, and plastics and polymers such as polyacrylamide, polystyrene and polycarbonate. However, it is also possible to use an electrode itself as described below without using a substrate.

  In the case of a nucleic acid probe-immobilized substrate for performing fluorescence detection, the nucleic acid probe may be immobilized on any of the above substrates via a spacer. In the case of a nucleic acid probe-immobilized substrate for electrochemical detection, an electrode is disposed on any of the above substrates so that electrochemical detection is possible, and the nucleic acid probe is immobilized on the electrode. You just have to.

The electrode that can be used in the present invention is not particularly limited. For example, carbon electrodes such as graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon fiber, platinum, platinum black, gold, palladium, Examples include noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide and lead oxide, semiconductor electrodes such as Si, Ge, ZnO, CdS, TiO ₂ and GaAs, and titanium. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with other surface treatment agents as desired.

  The nucleic acid probe may be fixed through the spacer by any means known per se. For example, the spacer may be fixed to the electrode, and then the nucleic acid probe may be further fixed to the spacer. Alternatively, a spacer may be bonded to the nucleic acid probe in advance and fixed to the electrode through the spacer. Alternatively, the spacer and the nucleic acid probe may be synthesized on the electrode by means known per se. In addition, the nucleic acid probe may be immobilized through a spacer by directly immobilizing the spacer on a treated or untreated substrate or electrode surface by covalent bonding, ionic bonding, physical adsorption, or the like. Alternatively, a linker agent that assists in immobilizing the nucleic acid probe via the spacer may be used, and the nucleic acid probe may be immobilized on the substrate or the electrode via the spacer using such a linker agent. Further, a blocking agent for preventing nonspecific binding of the sample nucleic acid to the electrode may be treated on the electrode together with the linker agent. Moreover, the linker agent and blocking agent used here may be substances for advantageously performing electrochemical detection, for example.

  In addition, each nucleic acid probe having a different base sequence may be immobilized on a different electrode via a spacer, and a plurality of nucleic acid probes having different base sequences may be mixed with one electrode. It may be fixed via a spacer.

5). Detection The nucleic acid probe-immobilized substrate according to the present invention is an electrochemical cell as a means for detecting the presence of a double strand resulting from a hybridization reaction between a nucleic acid probe immobilized on the substrate and a target nucleic acid. Methods and fluorescence detection methods can be utilized.

(1) Electrochemical detection Electrochemical detection of double-stranded nucleic acid may be performed using, for example, a known double-stranded recognition substance.

  The double-stranded recognizer used here is not particularly limited. For example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisacridine, trisintercalator and polyintercalator Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. In addition, any other known double-stranded recognition substance is preferably used in the present invention.

  In the nucleic acid probe-immobilized substrate according to the present invention, the nucleic acid probe is immobilized on the electrode via a spacer. In the detection of double-stranded nucleic acid using such an electrode, a counter electrode or a reference electrode may be used in the same manner as in other general electrochemical detection methods. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.

  For example, when detecting whether or not the target nucleic acid is contained in the sample nucleic acid, the test may be performed as follows. For example, a nucleic acid component is extracted as a sample nucleic acid from a sample collected from an individual such as an animal including a human, a tissue or a cell. The obtained sample nucleic acid is subjected to treatment such as reverse transcription, extension, amplification and / or enzyme treatment, as necessary. The pretreated sample nucleic acid is brought into contact with the nucleic acid probe immobilized on the nucleic acid probe-immobilized substrate, and the reaction is carried out under conditions that allow appropriate hybridization. Such appropriate conditions depend on various conditions such as the type of base contained in the target sequence, the type of spacer and nucleic acid probe provided on the nucleic acid probe-immobilized substrate, the type of sample nucleic acid, and the state thereof. If it is a trader, it can select suitably. Although not limited to this, for example, the reaction may be performed under the following conditions.

  That is, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The sample nucleic acid obtained here is added and heat denatured at 90 ° C. or higher. The nucleic acid probe-immobilized substrate may be inserted into the heat-denatured sample nucleic acid immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate.

  During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, the electrode is washed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used. When the target nucleic acid containing the target sequence is present in the sample nucleic acid, it hybridizes with the nucleic acid probe, thereby generating a double-stranded nucleic acid.

  Subsequently, the double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. In general, after the hybridization reaction, the substrate is washed, and a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and the resulting signal is electrochemically measured.

  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 may be used.

  For example, the electrochemical measurement may be performed by applying a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically and measuring the reaction current value derived from the double-stranded recognizer. At this time, the potential may be swept at a constant speed, applied with a pulse, or a constant potential may be applied. In the measurement, for example, the current and voltage may be controlled by using a device such as a potentiostat, a digital multimeter, and a function generator. For example, the concentration of the target nucleic acid may be calculated from a calibration curve based on the obtained current value.

  In addition, electrochemical detection means known per se, for example, means disclosed in the following documents (Hashimoto et al. 1994, Wang et al. 1998) can be preferably used in the method of the present invention. In this document, Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anode current derived from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. This biosensor configuration includes immobilization of an inosine displacement probe (without guanine) to a carbon paste electrode and chronopotentiometric detection of duplex formation due to the presence of the guanine oxidation peak of the label. The detection means described in these documents may preferably be used in the present invention.

(2) Fluorescence detection method In the case of a method using a fluorescent labeling substance, the sample nucleic acid is a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine, or an enzyme such as biotin, hapten, oxidase or phosphatase, or ferrocene or quinones. Etc., and labeled with an electrochemically active substance. Alternatively, detection is performed by using a second probe labeled with the aforementioned substance. A plurality of labeling substances may be used simultaneously.

  In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample substance and a probe immobilized on a probe-immobilized chip is performed, for example, as follows. That is, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The nucleic acid component extracted here is added and heat-denatured at 90 ° C. or higher. The probe-immobilized chip may be inserted immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, washing is performed. For the washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.

  In the case of fluorescence detection, the hybridization reaction is detected by detecting the labeled base sequence 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.

  Also within the scope of the present invention is a method for detecting the presence of any target nucleic acid or target sequence using the nucleic acid probe of the present invention as described above.

  In particular, a sample nucleic acid is amplified using a primer for obtaining the relationship between X and Y as described above, and the obtained amplification product is immobilized on a nucleic acid probe-immobilized substrate according to an embodiment of the present invention. It is possible to obtain better hybridization efficiency by detecting the presence of the target nucleic acid by reacting and detecting the resulting hybridization. Such a method is also included in the present invention. In addition, amplification that can be used in such a method uses, for example, reverse transcriptase even in amplification such as polymerase chain reaction (generally referred to as PCR, hereinafter referred to as PCR). Even reverse transcription PCR, such as reverse transcription amplification, includes any other amplification known per se.

  Amplifications that can be used according to embodiments of the present invention include, for example, Nucleic acid strand amplification (NASBA), Transcription mediated amplification (TMA), Ligase chain reaction (LCR), Strand displacement amplification (SDA), Isothermal and Chimeric primer-initiated Amplification of Nucleic Amplification methods such as acids (ICANN) and Rolling circle amplification (RCA).

  The nucleic acid analysis method according to the embodiment of the present invention includes analysis of a sample nucleic acid contained in a sample, for example, detection and quantification of the presence of a target sequence, expression analysis such as the disappearance of gene expression, single nucleotide polymorphism (Single Nucleotide) Analysis of polymorphism (ie, polymorphism, ie SNP) and microsatellite sequences, disease diagnosis and disease risk prediction by analysis of disease-related genes, detection of infection, virus type analysis, toxicity testing, etc. It can be used when Therefore, it can be used for various clinical purposes such as clinical diagnosis and onset prediction. Further, it can be widely used for various basic researches and applied researches such as food inspection, quarantine, pharmaceutical inspection, forensic medicine, agriculture, livestock, fishery and forestry.

  Examples of the nucleic acid detection method according to the present invention will be described below.

  In this example, the relationship between the length (X) of the spacer of the nucleic acid probe and the length (Y) from the nucleic acid probe binding site to the end on the substrate side in the target nucleic acid and the degree of hybridization efficiency were examined. It is a thing.

(1) Relationship between nucleic acid probe and target nucleic acid The relationship between the nucleic acid probe used in this example and the target nucleic acid is shown in FIG. Detailed sequences of the nucleic acid probe and the target nucleic acid will be described later. First, the general configuration and correlation thereof will be described.

  FIG. 4 shows the nucleic acid probe C-0, the nucleic acid probe C-10, the nucleic acid probe C-20, and the nucleic acid probe C-30 together with the target nucleic acid 70-0, the target nucleic acid 70-20, and the target nucleic acid 70-40. It is the figure written together on the basis of the target sequence which is 20 bases, and the sequence complementary to a target sequence.

  There are four types of nucleic acid probes used here. The sequence complementary to the target sequence of the nucleic acid probe is 20 bases and corresponds to the hatched portion in FIG.

  The nucleic acid probe C-0 has no spacer at the 5 'end.

  The nucleic acid probe C-10 has a spacer X1 composed of 10 bases of cytosine at the 5 'end.

  The nucleic acid probe C-20 has a spacer X2 comprising 20 bases of cytosine at the 5 'end.

  The nucleic acid probe C-30 is provided with a spacer X3 consisting of 30 bases of cytosine at the 5 'end. These four types of nucleic acid probes are the same except for the spacer. These nucleic acid probes are all immobilized on the substrate at the 5 'end.

  There are three types of target nucleic acids used here. The target sequences possessed by these three types of target nucleic acids are equally long and 20 bases. In FIG. 4, the target sequence corresponds to the shaded portion. The base sequences of the target sequences included in the three types of target nucleic acids are equal.

  The target nucleic acid 70-0 is a nucleic acid having a total length of 70 bases, and a target sequence of 20 bases is present at the 3 'end thereof.

  The target nucleic acid 70-20 is a nucleic acid having a total length of 70 bases. There are 30 bases on the 5 'side of the target sequence and a 20-base sequence Y1 on the 3' side of the target sequence.

  The target nucleic acid 70-40 is a nucleic acid having a total length of 70 bases. There are 10 bases on the 5 'side of the target sequence and a 40-base sequence Y2 on the 3' side of the target sequence.

(2) Nucleic acid probe The sequence complementary to the target sequence contained in the nucleic acid probe is 20 bases. Nucleic acid probes C-0, C-10, C-20, and C-30 are respectively composed of C (ie, cytosine) at 0 base, 10 base, 20 base, and 5 ′ end of the 20 base nucleic acid probe sequence. A probe added as a spacer with 30 bases. The sequence is as follows.

C-0: 5'-SH-TGGACGAAGACTGACGCTC-3 '(SEQ ID NO: 1)
C-10: 5′-SH- (C ₁₀ ) TGGACGAAGACTGACGCTC-3 ′ (SEQ ID NO: 2)
C-20: 5′-SH- (C ₂₀ ) TGGACGAAGACTGACGCTC-3 ′ (SEQ ID NO: 3)
C-30: 5'-SH- ( _C30 ) TGGACGAAGACTGACGCTC-3 '(SEQ ID NO: 4)
A thiol group is modified at the 5 ′ end of the above four types of probes, ie, C-0, C-10, C-20, and C-30. Moreover, X of the nucleic acid probes C-0, C-10, C-20, and C-30 is 0 base, 10 bases, 20 bases, and 30 bases, respectively.

(3) Target Nucleic Acid On the other hand, as a target nucleic acid model, a 70-base oligonucleotide containing a sequence complementary to the 20-base sequence was prepared. This oligonucleotide has three types of lengths from the end of the probe binding site to the 3 ′ end: 0 base, 20 bases and 40 bases. Each sequence is as shown below.

Target nucleic acid 70-0 (SEQ ID NO: 5):
5'CTATAAACATGCTTTCCGTGGCAGTGAGAACAAATGGGACCGTGCATTGC (GAGCGTCAGTCTTCGTCCAG)
Target nucleic acid 70-20 (SEQ ID NO: 6):
5'CTATAAACATGCTTTCCGTGGCAGTGAGAA (GAGCGTCAGTCTTCGTCCAG) CAAATGGGACCGTGCATTGC
Target nucleic acid 70-40 (SEQ ID NO: 7)
5 'CTATAAACAT (GAGCGTCAGTCTTCGTCCAG) GCTTTCCGTGGCAGTGAGAACAAATGGGACCGTGCATTGC
The sequence between parentheses “()” is the probe binding site, and the 5 ′ end is labeled with a fluorescent dye. These target nucleic acids Y in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 are 0 base, 20 base and 40 base, respectively.

(4) Immobilization of nucleic acid probe In this example, a gold substrate was used as a substrate. The gold substrate was immersed in a buffer solution containing each of the nucleic acid probes C-0, C-10, C-20, and C-30 and allowed to stand at room temperature for 1 hour. Thereafter, the nucleic acid probe-immobilized gold substrate was prepared by washing with distilled water and drying.

(5) Hybridization of target nucleic acid A buffer solution containing each of the three types of target nucleic acids was heat denatured at 95 ° C. for 5 minutes. Thereafter, it was rapidly cooled in ice to obtain a target nucleic acid solution. A nucleic acid probe-immobilized gold substrate on which each nucleic acid probe was immobilized was immersed in this target nucleic acid solution. This was left to stand at 35 ° C. for 1 hour. Thereafter, the nucleic acid probe-immobilized gold substrate was immersed in a buffer solution containing no nucleic acid and allowed to stand at 35 ° C. for 1 hour for washing.

(6) Detection of hybridized target nucleic acid By detecting the fluorescence intensity derived from the fluorescent dye modified at the 5 ′ end of the target nucleic acid, the presence of the target nucleic acid hybridized to the immobilized nucleic acid probe is detected. Detected.

(7) Results (i) When using the target nucleic acid 70-0 The results when using the target nucleic acid 70-0 are shown in FIG. FIG. 6A schematically shows how the target nucleic acid 70-0 and the nucleic acid probes C-0, C-10, C-20, and C-30 are bound. In any nucleic acid probe, X ≧ Y. At this time, as shown in FIG. 6B, the amount of the target nucleic acid 70-0 hybridized with each of the nucleic acid probe C-0, the nucleic acid probe C-10, the nucleic acid probe C-20, and the nucleic acid probe C-30 is almost the same. It was found from the fluorescence intensity detected to be comparable.

(Ii) When target nucleic acid 70-20 is used The result when target nucleic acid 70-20 is used is shown in FIG. FIG. 7A schematically shows the state of binding between the target nucleic acid 70-20 and the nucleic acid probes C-0, C-10, C-20, and C-30. In the nucleic acid probes C-0 and C-10, X <Y, in the nucleic acid probe C-20, X = Y, and in the nucleic acid probe C-30, X> Y.

  At this time, the detected fluorescence intensity became stronger depending on the length of the spacer of the nucleic acid probe, as shown in FIG. 7B. Similarly, depending on the length of the spacer, the amount of hybridization of the target nucleic acid 70-20 increases, and cytosine 20 bases (ie, C20) equal to the number of bases from the nucleic acid probe binding site to the 3 ′ end of the target nucleic acid. ) Was the maximum when using a nucleic acid probe containing a spacer, and was almost the same as that when using a spacer containing more bases (FIG. 7B).

(Iii) When target nucleic acid 70-40 is used When target nucleic acid 70-40 is used as the target nucleic acid, binding of target nucleic acid 70-20 and nucleic acid probes C-0, C-10, C-20 and C-30 This is schematically shown in FIG. 8A. In any nucleic acid probe, X <Y. At this time, the target nucleic acid hybridized with each nucleic acid probe was almost the same in any case, and the hybridization efficiency was low (FIG. 8B).

(Iv) Summary From the above results, the length (X) of the spacer used when binding the nucleic acid probe to the solid phase carrier and the target nucleic acid containing the target sequence as a part of the nucleic acid probe were hybridized. In this case, it was confirmed that the hybridization efficiency was improved when the relationship X ≧ Y was established between the length (Y) from the substrate-side end of the hybridization site to the substrate-side end of the target nucleic acid. . By improving the hybridization efficiency, the presence of the target nucleic acid can be detected with higher accuracy.

(8) Regulation 1 by amplification of sample nucleic acid using primers
According to a further aspect of the present invention, a sample nucleic acid is prepared using a nucleic acid probe that provides a preferred target nucleic acid before reacting the nucleic acid probe-immobilized substrate with the sample nucleic acid according to the above-described aspect of the present invention. A method comprising the step of amplifying is provided. Such a nucleic acid probe has a target nucleic acid within about 40 bases, preferably about 26 bases to about 12 bases from the end of the substrate side of the target sequence site when the target nucleic acid hybridizes with the nucleic acid probe with the target sequence. A primer for amplifying a sample nucleic acid may be used so that the end of the nucleic acid is located.

  FIG. 9 is a diagram showing the relationship between an amplified fragment and a nucleic acid probe according to a further embodiment of the present invention. Here, a system for detecting the MxA gene in the human genome is shown. FIG. 9 shows the nucleic acid probe of SEQ ID NO: 8 and two types of PCR products. The nucleic acid probe of SEQ ID NO: 8 has a spacer portion of 20 bases (denoted as “Spacer-20” in the figure). A PCR product A (hereinafter referred to as “A”) was prepared using a primer represented by the base sequence of SEQ ID NO: 9 in which the 5 ′ end was biotinylated and a primer represented by the base sequence of SEQ ID NO: 10 labeled with cy5. A PCR product B (hereinafter referred to as “B”) was prepared using a primer represented by the base sequence of SEQ ID NO: 11 with the 5 ′ end biotinylated and a primer represented by the base sequence of SEQ ID NO: 12 labeled with cy5. Single strands were prepared using avidin-labeled magnetic microparticles. In FIG. 9, A is 12 bases (referred to as “12mer” in the figure) from the end of the nucleic acid probe binding site, and B is 26 bases (referred to as “26mer” in the figure). A hybridization reaction was performed using this target, and the fluorescence intensity was measured. As a result, A showed a fluorescence intensity about 10 times that of B (FIG. 10).

  Further, the reaction of B reached saturation in about 1 hour, whereas A reached saturation in about 10 minutes. Furthermore, as a result of examining the specificity of SNP detection, the S / N ratio between A and B was about twice different.

(9) Regulation by amplification of sample nucleic acid using primers 2
FIG. 11 is a diagram showing the relationship between amplified fragments and probes according to a further embodiment of the present invention. In FIG. 11, the result of the example of the amplification product obtained by amplifying in order to determine the polymorphism of the MBL gene in a human genome, and a nucleic acid probe is shown. FIG. 11 shows the nucleic acid probe represented by the nucleotide sequence of SEQ ID NO: 13 and three kinds of PCR products. A PCR product C (hereinafter referred to as “C”) was prepared using a primer represented by the nucleotide sequence of SEQ ID NO: 14 phosphorylated at the 5 ′ end and a primer represented by the nucleotide sequence of SEQ ID NO: 15 labeled with cy5. A PCR product D (hereinafter referred to as “D”) was prepared using a primer represented by the base sequence of SEQ ID NO: 16 phosphorylated at the 5 ′ end and a primer represented by the base sequence of SEQ ID NO: 15 labeled with cy5. The PCR product E (hereinafter referred to as “E”) was prepared using a primer represented by the nucleotide sequence of SEQ ID NO: 18 phosphorylated at the 5 ′ end and a primer represented by the nucleotide sequence of SEQ ID NO: 17 labeled with cy5. Furthermore, a primer was used so that the distance from the end of the nucleic acid probe binding site of the PCR product was 40 bases. Single strand preparation was performed using λ nuclease. As shown in FIG. 11, in the PCR products, C is 13 bases from the end of the probe binding site (indicated as “13mer” in the figure) and D is 33 bases (indicated as “33mer” in the figure). , E is 48 bases (denoted as “48mer” in the figure). Although not shown, a PCR product having a distance of 40 bases from the end of the probe binding site was also obtained. A hybridization reaction was performed using these targets, and the fluorescence intensity was measured. As a result, C showed a fluorescence intensity about 6 times that of D and about 40 times that of E (FIG. 12).

  FIG. 13 shows the detection result of the MBL detection system by an electrochemical method. After hybridization reaction with each target, Hoechst 33258 current was measured. As a result, the target C showed a current value about 3 times greater than D and 10 times greater than E.

  From the above results, it was found that when the 3 'end of the primer of 40 bases or more from the center of the site to which the nucleic acid probe binds is located at a site separated from the center, the hybridization efficiency is greatly reduced. Furthermore, since a phenomenon that the specificity also decreases was observed, in order to detect a nucleic acid with high speed, high selectivity, and high sensitivity, a primer located within 40 bases from the center of the site to which the nucleic acid probe was bound was used. It is important to amplify.

  According to the present invention as described above, a method for detecting a nucleic acid sequence having high detection sensitivity and specificity and a primer used for the method are provided.

  Additional advantages and modifications will be readily found by those skilled in the art. Accordingly, the invention in its broader aspects is not limited to the detailed description and exemplary embodiments presented herein. Accordingly, various modifications can be made without departing from the spirit or scope of the general inventive idea as defined by the appended claims and their equivalents.

FIG. 1A is a plan view showing an example of a nucleic acid probe-immobilized substrate of the present invention, and FIG. 1B is a cross-sectional view taken along line BB in FIG. 1A. The figure which shows an example of the nucleic acid probe fixed base | substrate of this invention Diagram showing binding state of nucleic acid probe and target nucleic acid The figure which shows typically the nucleic acid probe and target nucleic acid which were used in the Example. Graph showing the relationship between X and Y The figure which shows the binding state of the nucleic acid probe containing various spacers used in the examples and the target nucleic acid (FIG. 6A), and the figure which shows the results obtained by the tests performed in the examples (FIG. 6B) The figure which shows the binding state of the nucleic acid probe containing various spacers used in the examples and the target nucleic acid (FIG. 7A), and the figure which shows the results obtained by the tests conducted in the examples (FIG. 7B) The figure which shows the binding state of the nucleic acid probe containing various spacers used in the examples and the target nucleic acid (FIG. 8A), and the figure which shows the results obtained by the tests performed in the examples (FIG. 8B) The figure which shows the relationship between the amplified fragment used in the Example and a nucleic acid probe The graph which shows the result obtained by the test done in the Example The figure which shows the relationship between the amplified fragment used in the Example and a nucleic acid probe The graph which shows the result obtained by the test done in the Example The graph which shows the result obtained by the test done in the Example

Explanation of symbols

1, 11, 30 Nucleic acid probe-immobilized substrate; 2, 12, 31 substrate; 3, 32 electrodes; 4, 13, 34 spacers; 5, 14, 35 nucleic acid probe; 6 pads; 33a linker agent; 33b blocking agent; Target nucleic acid

Claims (7)

A nucleic acid probe-immobilized substrate having an electrode capable of performing electrochemical detection and having a nucleic acid probe immobilized on the electrode via a spacer, wherein the length of the spacer is X, and the nucleic acid probe When a target nucleic acid having a total length of 70 bases or more including a base sequence complementary to is hybridized to the nucleic acid probe, the length from the substrate-side end of the hybridization site to the substrate-side end of the target nucleic acid is represented by Y A method of detecting the presence of a target nucleic acid in a sample nucleic acid using a nucleic acid probe-immobilized substrate in which the relationship of X ≧ Y, Y ≧ 10Å, and 200Å ≧ X is established,
The sample nucleic acid is amplified using the selected primers so that the relationship between X and Y in the sample nucleic acid satisfies X ≧ Y, Y ≧ 10Å, and 200Å ≧ X, and the total length is 70 bases or more. Process,
Reacting the amplification product obtained by the amplification with the nucleic acid probe immobilized on the nucleic acid probe-immobilized substrate under conditions that allow appropriate hybridization;
A method for detecting the presence of the target nucleic acid, comprising: electrochemically detecting the hybridization produced by the reacting step and determining that the target nucleic acid is present in the sample nucleic acid. A nucleic acid probe-immobilized substrate having an electrode capable of performing electrochemical detection and having a nucleic acid probe immobilized on the electrode via a spacer, wherein the length of the spacer is X, and the nucleic acid probe When a target nucleic acid having a total length of 70 bases or more including a base sequence complementary to is hybridized to the nucleic acid probe, the length from the substrate-side end of the hybridization site to the substrate-side end of the target nucleic acid is represented by Y In such a case, a method for detecting the presence of a target nucleic acid having a total length of 70 bases or more including the target sequence using a nucleic acid probe-immobilized substrate in which the relationship X ≧ Y, Y ≧ 10Å, and 200Å ≧ X is established. There,
(a) When the target nucleic acid is hybridized with the nucleic acid probe in a target sequence, the target nucleic acid is positioned so that the base end of the target nucleic acid is located within 40 bases from the base end of the target sequence site. Preparing primers for amplifying nucleic acids;
(b) amplifying a sample nucleic acid using the primer prepared in (a) above;
(c) making the amplification product obtained in (b) single-stranded,
(d) reacting the single strand obtained in (c) with the nucleic acid probe;
(e) A method for detecting the presence of a target nucleic acid, comprising detecting the presence of the target nucleic acid in the sample nucleic acid by electrochemically detecting the hybridization produced in (d).   3. The method for detecting the presence of a target nucleic acid according to claim 1 or 2, wherein the relationship between X and Y is X ≧ Y, Y ≧ 10Å, and X−10Å ≧ Y.   The target nucleic acid according to claim 1 or 2, wherein the relationship between X and Y is X≥Y, Y≥10 ≧, X-10Å≥Y, 200Å≥X, and X≥100Å How to detect the presence of   3. The presence of the target nucleic acid according to claim 1 or 2, wherein the relationship between X and Y is X ≧ Y, Y ≧ 10Å, X-10Å ≧ Y, and 100Å ≧ X. Method. The method for detecting the presence of a target nucleic acid according to any one of claims 1 to 5 , wherein the spacer is an organic chain molecule. The method for detecting the presence of a target nucleic acid according to any one of claims 1 to 5 , wherein the spacer is selected from the group consisting of a nucleic acid, ethylene glycol, and alkane.

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