JP2004073064A - Nucleic acid fragment, nucleic acid probe immobilized substrate and assay kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for simply detecting and/or elongating a nucleic acid chain in high sensitivity. <P>SOLUTION: The nucleic acid fragment is a nucleic acid fragment which is hybridized with a target nucleic acid containing a first target sequence x1 and a second target sequence x2 and comprises a sequence X1 complementary to the target sequence x1, a sequence Y, a sequence Z, a sequence y complementary to the sequence Y and a sequence X2 complementary to the target sequence x2 and is represented by X1-Y-Z-y-X2. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】本発明の態様に従う核酸断片の１例の概略構成を示す図。
【図２】実施例１および２に係る核酸プローブ固定化基体の概略構成を示す平面図。
【図３】実施例３および４に係る核酸プローブ固定化基体の概略構成を示す平面図。
【図４】実施例４に係る比較実験の結果を示すグラフ。
【符号の説明】
１．基体　　２．固定化領域　　１１．基体　　１２．電極　　１３．パット[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a nucleic acid fragment, a nucleic acid probe-immobilized substrate, and an assay kit.
[0002]
[Prior art]
In order to detect a nucleic acid strand having a specific nucleic acid sequence, first, amplification is performed using a pair of nucleic acid primers complementary to the sequence to be detected, followed by electrophoresis or complementary to the sequence to be detected. A method of confirming or detecting an amplification product using a substrate or the like on which a typical nucleic acid probe is immobilized is generally used (Japanese Patent No. 2573443 and Japanese Patent No. 3171793). In that case, in order to specifically detect a specific nucleic acid strand, a probe or primer having a continuous specific sequence of about 15 bases or more is required. However, even with the use of primer or probe design software, it is not always easy to select a 15-base continuous specific sequence in a region with many mutations and polymorphisms. Therefore, there is a problem that an optimal detection condition cannot be set.
[0003]
On the other hand, a current detection type DNA chip using a probe-immobilized electrode and an electrochemically active intercalator has been reported. This method has advantages in that running costs are low because no label is required, and that an expensive and large detection device such as in the case of fluorescence detection is not required. In such electrochemical detection, it is considered that a longer nucleic acid probe has higher detection sensitivity, but the intercalating agent slightly reacts with the single-stranded probe itself. Therefore, if a long probe is used, noise increases, and the detection sensitivity is insufficient.
[0004]
In addition, when performing a specific reaction using a long probe, the melting temperature (Tm) becomes high. Therefore, harmful additives such as dimethylsulfoxide (DMSO) and dimethylformamide are added to lower the temperature. There is a need to.
[0005]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a means that enables simple and highly sensitive detection and / or extension of a nucleic acid chain.
[0006]
[Means for Solving the Problems]
The above object is achieved by the present invention as described below. That is,
A nucleic acid fragment for hybridizing to a target nucleic acid containing a first target sequence x1 and a second target sequence x2, wherein the sequence X1 is complementary to the target sequence x1, an arbitrary sequence Y, an arbitrary sequence Z, A nucleic acid fragment comprising a sequence y complementary to the sequence Y and a sequence X2 complementary to the target sequence x2, and represented by X1-YZyX2;
A nucleic acid fragment to be hybridized to a target nucleic acid containing a sequence from the first target sequence x1 to the (n + 1) th target sequence xn + 1, wherein the sequence X1, the optional sequence Y1, and the optional sequence complementary to the target sequence x1 From X1-Y1-Z1-y1 consisting of Z1 and the sequence y1 complementary to the sequence Y1, a sequence Xn complementary to the target sequence xn, an arbitrary sequence Yn, an arbitrary sequence Zn, and a sequence complementary to the sequence Yn yn and a sequence Xn-Yn-Zn-yn and a sequence Xn + 1 complementary to the target sequence xn + 1, and X1-Y1-Z1-y1... -Xn-Yn-Zn-yn-Xn + 1. (Where n is an integer of 2 or more).
[0007]
An assay kit comprising any one of the above nucleic acid fragments as a reagent selected from the group consisting of a primer and a nucleic acid probe;
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 A nucleic acid probe for detecting a human hepatitis C virus represented by a nucleotide sequence;
A primer for amplifying a nucleic acid derived from human hepatitis C virus represented by the base sequence described in the sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26;
It is.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
1. Nucleic acid fragments
The nucleic acid fragment according to the embodiment of the present invention is a nucleic acid fragment to be used after being hybridized to a target sequence with a partial sequence thereof. Hereinafter, the nucleic acid fragment according to the embodiment of the present invention will be described using one example shown in FIG.
[0009]
The target nucleic acid to which the nucleic acid fragment according to the embodiment of the present invention hybridizes includes a target sequence x1 and a target sequence x2. On the other hand, the nucleic acid fragment according to the embodiment of the present invention includes a sequence X1 complementary to the target sequence x1 and a sequence X2 complementary to the target sequence x2, between which an arbitrary sequence Y and a sequence complementary thereto. y and any sequence Z between them. That is, the nucleic acid fragment is represented by the general formula “X1-YZZyX2”.
[0010]
Here, the sequences “x1” and “x2” (here, x is a lowercase letter) included in the target sequence, and the sequences “X1” and “X2” (here, X is an uppercase letter) contained in the nucleic acid fragment, respectively. Complementary to each other. The sequence “y” (where y is lowercase) and the sequence “Y” (where Y is uppercase) contained in the nucleic acid fragment are complementary to each other. In the present specification, for the sake of convenience, when the same alphabet is shown in uppercase and lowercase, it indicates that they are sequences consisting of base sequences complementary to each other.
[0011]
As used herein, the term "target sequence" refers to a base sequence to which a nucleic acid fragment according to an embodiment of the present invention binds. As used herein, the term "target nucleic acid" refers to a nucleic acid that includes a target sequence.
[0012]
As used herein, the term "nucleic acid" refers to any nucleic acid analog, such as DNA and RNA, S-oligos, methyl phosphonate oligos, any nucleic acid analog such as PNA (i.e., peptide nucleic acids) and LNA (i.e., locked nucleic acids). The term is a general term for a substance whose partial structure can be represented by a base sequence. Such a nucleic acid may be a naturally occurring one or an artificially synthesized one.
[0013]
The nucleic acid fragment of the present invention may be generally synthesized or produced by a method known per se. For example, such a method may use a nucleic acid synthesizer used for general nucleic acid synthesis, or may use a means such as general genetic manipulation using Escherichia coli or the like. The synthesized or produced nucleic acid is desirably purified by liquid chromatography, mass spectroscopy, or the like.
[0014]
The terms “complementary,” “complementary,” and “complementary,” as used herein, indicate that the sequences are 50% to 100% complementary to each other. In the present invention, the complementarity is preferably 80% or more, more preferably 90% or more. The terms "homologous", "homologous" and "homology" as used herein indicate that a plurality of sequences are homologous to each other in the range of 50% to 100%. In the present invention, the homology is preferably 80% or more of homology, more preferably 90% or more of homology.
[0015]
The length of each of the sequence X1 and the sequence X2 is not particularly limited, but may be in the range of about 5 bases to about 50 bases, preferably about 5 bases to about 9 bases.
[0016]
The length of each of the sequence Y and the sequence y is not particularly limited, but may be in the range of about 3 bases to about 50 bases, preferably about 5 bases to about 10 bases.
[0017]
The complementarity between the sequence Y and the sequence y is preferably 80%, more preferably 90% or more. The higher the complementarity between sequence Y and sequence y, the greater the stability when bound to the target sequence.
[0018]
The length of sequence Z is not particularly limited, but may be about 3 to about 50 bases, and preferably about 4 to about 10 bases.
[0019]
Conventionally, specific detection and amplification of a nucleic acid usually requires a continuous sequence of about 15 bases or longer. However, in the case of the nucleic acid fragment disclosed by the present invention, the target sequence can be specifically detected, amplified and extended even if it is a continuous sequence of 9 bases or less.
[0020]
Further, the nucleic acid fragment according to the present invention has a sequence Y, a sequence y, and a sequence Z that are not involved in hybridization with the target sequence. By having these sequences, a double-stranded structure, that is, a hairpin structure, is obtained in the nucleic acid fragment.
[0021]
In the above-described example, an example in which one hairpin structure in the nucleic acid fragment can be included has been described. However, the present invention is not limited thereto, and a plurality of hairpin structures may be included in one nucleic acid fragment. In that case, it is preferable that a sequence complementary to the target sequence exists between adjacent hairpin structures.
[0022]
For example, in the case of a nucleic acid fragment for hybridizing to a target nucleic acid containing a sequence from the first target sequence x1 to the (n + 1) th target sequence xn + 1, a sequence X1 complementary to the first target sequence x1, a sequence Y1 X1-Y1-Z1-y1 consisting of a sequence Z1 and a sequence y1 complementary to the sequence Y1, a sequence Xn complementary to the n-th target sequence xn, a sequence Yn, a sequence Zn, and A nucleic acid fragment comprising a continuous sequence up to Xn-Yn-Zn-yn consisting of a sequence yn complementary to the sequence Yn and further including a sequence X (n + 1) complementary to the (n + 1) th target sequence xn + 1, , X1-Y1-Z1-y1-... -Xn-Yn-Zn-yn-Xn + 1. Here, n is an integer of 2 or more. Further, the arrangements of the arrangements Y1 to Yn may be the same arrangements, may be different arrangements, or may be partly equal arrangements.
[0023]
The nucleic acid fragment according to the embodiment of the present invention as described above is used to extend a complementary strand to a target nucleic acid containing the target sequence, or to amplify the target nucleic acid and its complementary strand. Can be preferably used as a primer.
[0024]
The target sequence or target nucleic acid to be detected by such a nucleic acid probe or amplified by a primer may be any type of nucleic acid, and may be the target of detection or amplification of the target sequence and / or target nucleic acid. Any nucleic acid sample containing such a nucleic acid may be used.
[0025]
As used herein, the term "nucleic acid sample" refers to a sample containing nucleic acids. A nucleic acid sample is a sample collected from an individual, for example, blood such as peripheral venous blood, leukocytes, serum, urine, feces, semen, saliva, cells, organs and tissues, and cultured cells. It may be prepared by a method known per se. The term "individual" as used herein may include humans, non-human animals and plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas. Further, a sample containing an artificially synthesized nucleic acid may be used. The sample may be subjected to any necessary pretreatment such as homogenate and extraction, if necessary. Such a pretreatment can be selected by those skilled in the art depending on the sample.
[0026]
2. Nucleic acid probe
A nucleic acid probe according to an embodiment of the present invention is used as a means for detecting a target sequence. That is, the nucleic acid probe has a sequence complementary to the target sequence to be detected. Therefore, it is possible to hybridize to the target sequence. Thereafter, by detecting the resulting hybridization, it is possible to detect the presence of the target sequence and / or the target nucleic acid containing the target sequence in the sample nucleic acid.
[0027]
As used herein, the term "detecting hybridization" refers to detecting double-stranded nucleic acid generated by hybridization, or pre-labeling a sample nucleic acid with any labeling substance, and after hybridization. This is a general term for detecting a signal derived from the labeling substance, or detecting, by other means known per se, the presence of double-stranded nucleic acid or the occurrence of hybridization by the reaction. Yes, detection may be achieved by any means.
[0028]
A nucleic acid probe according to an embodiment of the invention may comprise further sequences in addition to the nucleic acid fragment according to the invention described above. In addition, one of a fluorescent substance such as Cy5 and Cy3, a dye substance, a magnetic substance, and a pair of substances that specifically bind to each other such as avidin and biotin may be provided as a labeling substance. Further, an enzyme such as hapten, oxidase or phosphatase, or ferrocene or quinones may be provided. Such modified nucleic acid probes are also provided as the present invention.
[0029]
Further, the nucleic acid probe according to the present invention may be used by being immobilized on a substrate. Such a nucleic acid probe-immobilized substrate is also provided as an aspect of the present invention.
[0030]
3. Nucleic acid probe-immobilized substrate
The nucleic acid probe-immobilized substrate that can be provided in the embodiment of the present invention basically comprises a substrate and a nucleic acid probe immobilized on the substrate. Preferably, the nucleic acid probe is immobilized on the substrate. Further, a nucleic acid probe-immobilized substrate provided with the nucleic acid probe is also provided as an aspect of the present invention.
[0031]
As the nucleic acid probe-immobilized substrate, a device known per se, which is generally called a DNA chip or a DNA microarray or a DNA macroarray, may be used. In addition, microbeads, microtiter plates, and the like can be used as a probe-immobilized substrate.
[0032]
Here, the “nucleic acid probe” is a nucleic acid containing a base sequence complementary to a target sequence, and refers to a nucleic acid fragment to be immobilized on a substrate. The nucleic acid probe has a sequence that is complementary to the target sequence of interest, and thus can hybridize with the target sequence under appropriate conditions.
[0033]
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 called a target nucleic acid. For example, when the base sequence of the tag provided in the primer is used as the target sequence, the sequence of the nucleic acid probe may be a sequence complementary to the sequence of the desired tag.
[0034]
As used herein, the terms "complementary", "complementary", and "complementary" may be in the range of 50% to 100%, preferably 100% complementary. As used herein, the terms "homology", "homologous" and "homology" may be 50% to 100% homologous, preferably 100% homologous.
[0035]
The nucleic acid probe-immobilized substrate used in the embodiment of the present invention will be described below using examples.
[0036]
(1) First example
FIG. 2 schematically shows a first example of a nucleic acid probe-immobilized substrate that can be used according to the embodiment of the present invention. A nucleic acid probe-immobilized substrate as a first example includes a substrate 1 and a nucleic acid probe immobilized on an immobilized region 2 thereof (FIG. 2).
[0037]
Such a nucleic acid probe-immobilized substrate can be produced, for example, by immobilizing the nucleic acid probe on a substrate such as a silicon substrate by a means known per se.
[0038]
In this embodiment, the number of nucleic acid probes arranged on one substrate is not limited to this, and may be changed as desired, and a nucleic acid probe having a plurality of types of base sequences may be added to one substrate. It may be arranged. The solid phase pattern of a plurality and / or a plurality of types of nucleic acid probes on a substrate can be appropriately changed by a person skilled in the art as needed. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.
[0039]
(2) Second example
A second example of the nucleic acid probe-immobilized substrate that can be used in the embodiment of the present invention will be described with reference to FIG. The nucleic acid probe-immobilized substrate as the second example includes a nucleic acid probe immobilized on an electrode 12 provided on a substrate 11 (FIG. 3). The electrode 12 is connected to a pad 13 for extracting electrical information.
[0040]
Such a nucleic acid probe-immobilized substrate can be produced, for example, by arranging electrodes on a substrate such as a silicon substrate by means known per se, and immobilizing the nucleic acid probe on the electrode surface. .
[0041]
In the present embodiment, the number of electrodes is set to 10, but the number of electrodes arranged on one base is not limited to this. Further, the arrangement pattern of the electrodes is not limited to the pattern shown in FIG. 3, and a person skilled in the art can appropriately change the design as needed. A reference electrode and a counter electrode may be provided as necessary. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.
[0042]
In the case of a nucleic acid probe-immobilized substrate for performing fluorescence detection as described in the above example, the nucleic acid probe may be immobilized on any of the above substrates. Further, in the case of a nucleic acid probe-immobilized substrate for performing electrochemical detection as described in the first example, an electrode is attached to any of the substrates so that electrochemical detection is possible. It is sufficient to dispose and immobilize the nucleic acid probe on the electrode.
[0043]
Electrodes that can be used in the present invention are not particularly limited, for example, graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon electrode such as carbon fiber, platinum, platinum black, gold, palladium, Noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide and lead oxide, Si, Ge, ZnO, CdS, TiO ₂ , A semiconductor electrode such as GaAs, and titanium. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with another surface treating agent as desired.
[0044]
The nucleic acid probe may be fixed by any means known per se. For example, the nucleic acid probe may be directly immobilized 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 may be used, and the nucleic acid probe may be immobilized on a substrate or an electrode via a spacer. Further, a blocking agent for preventing non-specific binding of the sample nucleic acid to the electrode may be treated on the electrode together with a linker agent. Further, the linker agent and the blocking agent used here may be, for example, substances for performing electrochemical detection advantageously.
[0045]
Further, nucleic acid probes having different base sequences may be respectively immobilized on different electrodes, and a plurality of types of nucleic acid probes having different base sequences are immobilized on one electrode in a mixed state. You may.
[0046]
(3) Detection
The nucleic acid probe-immobilized substrate according to the present invention is provided by an electrochemical method as a means for detecting the presence of a double strand resulting from a hybridization reaction between the nucleic acid probe immobilized on the substrate and a target nucleic acid. And fluorescence detection methods are available.
[0047]
(A) Electrochemical detection
The double-stranded nucleic acid can be detected electrochemically using, for example, a double-stranded recognition substance known per se.
[0048]
The double-stranded recognizer used here is not particularly limited, but for example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalators, and bisacridines, tris intercalators and polyintercalators Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene, viologen, or the like. Further, any other known double-stranded recognition substance is preferably used in the present invention.
[0049]
In the nucleic acid probe-immobilized substrate according to the present invention, the nucleic acid probe is immobilized on the electrode. Detection of a double-stranded nucleic acid using such an electrode may further use a counter electrode or a reference electrode, as in other general electrochemical detection methods. When a reference electrode is provided, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.
[0050]
Subsequently, the reaction is performed under conditions that allow appropriate hybridization. Such appropriate conditions can be determined by those skilled in the art depending on various conditions such as the type of base contained in the target sequence, the type of nucleic acid probe provided on the nucleic acid probe-immobilized substrate, the type of sample nucleic acid and their state. If so, it can be appropriately selected. Although not limited thereto, for example, the reaction may be performed under the following conditions.
[0051]
That is, the hybridization reaction solution is performed in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The sample nucleic acid obtained here is added and denatured at 90 ° C. or higher. Insertion of the nucleic acid probe-immobilized substrate into the heat-denatured sample nucleic acid may be performed immediately after denaturation or after quenching to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a substrate.
[0052]
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 about 1 night. After the hybridization reaction, the electrodes are 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. If 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.
[0053]
Subsequently, double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. Generally, after the hybridization reaction, the substrate is washed, a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and a signal generated thereby is electrochemically measured.
[0054]
The concentration of the double-stranded recognizer varies depending on its type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.
[0055]
For example, in the electrochemical measurement, a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically may be applied, and the reaction current value derived from the double-stranded recognizer may be measured. At this time, the potential may be swept at a constant speed, applied as a pulse, or a constant potential may be applied. At the time of measurement, for example, the current and voltage may be controlled 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.
[0056]
In such an electrochemical nucleic acid detection method, by using a nucleic acid probe having a hairpin structure having a hairpin structure according to an embodiment of the present invention (also referred to as a hairpin probe), an electrochemical agent using an intercalator is used. High sensitivity of the nucleic acid detection method is achieved. That is, before the hybridization reaction, the nucleic acid probe usually forms a single-stranded structure. However, by binding to, ie, hybridizing with, the target sequence, a double-stranded structure is formed between the target sequence and the nucleic acid probe, and a secondary structure (ie, double-stranded ) Is formed. Such a structure allows more intercalating agents to bind than those where the probe does not form a hairpin structure. As a result, an increase in current signal and an increase in sensitivity are achieved.
[0057]
Further, according to the embodiment of the present invention, the Tm can be set at a low temperature while maintaining the specificity, so that the hybridization reaction can be achieved at a low temperature without adding a special additional reagent. It is. It is possible to perform a PCR reaction.
[0058]
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 also be preferably used in the method of the present invention. In that article, 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 from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. The configuration of this biosensor involves immobilization of an inosine-substituted probe (without guanine) on 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.
[0059]
(B) Fluorescence detection method
In the case of using a fluorescent labeling substance, the antisense primer may be labeled with a substance capable of obtaining a fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Alternatively, detection may be performed by using a second probe labeled with the above-mentioned substance instead of the label. A plurality of labeling substances may be used simultaneously.
[0060]
In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample and a nucleic acid probe immobilized on a nucleic acid probe-immobilized chip is performed, for example, as follows. That is, the hybridization reaction solution is performed in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The extracted nucleic acid component is added thereto, and denatured at 90 ° C. or higher. Insertion of the nucleic acid probe-immobilized chip may be performed immediately after denaturation or after rapid cooling to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a 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 about 1 night. After the hybridization reaction, washing is performed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.
[0061]
In the case of fluorescence detection, the detection of the hybridization reaction is performed by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device according to the type of the label. When the label is a fluorescent substance, the label may be detected using, for example, a fluorescence detector.
[0062]
Use of the nucleic acid primer according to the present invention makes it possible to set Tm to a low temperature while maintaining specificity. Therefore, it is possible to achieve a hybridization reaction at a low temperature without adding a special additional reagent. It is possible to perform a PCR reaction.
[0063]
A method for detecting a target nucleic acid using the nucleic acid fragment, nucleic acid probe and / or nucleic acid probe-immobilized substrate of the present invention is also provided as an aspect of the present invention.
[0064]
4. Primer
A nucleic acid fragment according to an embodiment of the present invention may be used as a primer for hybridizing to a target sequence contained in a target nucleic acid to extend the nucleic acid.
[0065]
As used herein, the term "extend a nucleic acid" refers to extending a nucleic acid of interest by causing a polymerase to act using a template nucleic acid and a primer complementary to a partial base sequence thereof. And further indicates both such extension and amplification in which such extension is performed repeatedly.
[0066]
The extension reaction that may be utilized in accordance with aspects of the present invention may be the means commonly used to extend nucleic acids, known per se, or the means used to amplify nucleic acids. Any of such means may be used. Although not limited thereto, for example, a method utilizing a polymerase chain reaction (generally called PCR, hereinafter referred to as PCR), reverse transcription PCR, or the like can be used. Furthermore, Nucleic acid strand amplification (NASBA), Transcription mediated amplification (TMA), Ligase chain reaction (LCR), Strand displacement amplification (SDA), Isothermaland Chimeric primer-initiated Amplification of Nucleic acids (ICANN) and Rolling circle amplification (RCA Means) can also be used in accordance with aspects of the present invention.
[0067]
As used herein, "conditions under which an appropriate extension reaction can be obtained" refers to various conditions for the polymerase to synthesize a nucleic acid chain, for example, conditions such as temperature, pH, salt concentration, and dNTP, which make the activity of the polymerase appropriate. Indicate a condition that is sufficient to obtain
[0068]
Furthermore, a tag consisting of a base sequence, a fluorescent substance such as Cy5 and Cy3, a dye substance, a magnetic substance, and a pair of substances that specifically bind to each other such as avidin and biotin are added to the 5 ′ end of the primer. One may be provided as a labeling substance. Further, an enzyme such as hapten, oxidase or phosphatase, or ferrocene or quinones may be provided. Primers with such modifications are also provided as embodiments of the present invention.
[0069]
Use of the nucleic acid primer according to the present invention makes it possible to set Tm to a low temperature while maintaining specificity. Therefore, it is possible to achieve an extension reaction at a low temperature without adding a special additional reagent.
[0070]
5. Assay kit
In the present invention, the primer, the nucleic acid probe, and the nucleic acid probe-immobilized substrate according to any of the above-described embodiments of the present invention may be provided as an appropriate kit depending on the purpose of use.
[0071]
For example, in the case of a primer according to the present invention, a kit may be provided in combination with a reaction vessel and / or salts for buffer solution and / or other necessary reagents such as a polymerase and / or a substrate. For example, a nucleic acid probe containing a sequence complementary to a tag sequence, a primer and an extension product or a part thereof is also combined with a substrate and / or various reagents and / or labeling substances to form a kit. May be provided. The primers and the desired nucleic acid probe and / or nucleic acid probe-immobilized substrate may be combined as needed to provide a kit. However, the kit provided by the present invention is not limited to the above combinations, and may be provided as a kit in combination with various ones as necessary. The purpose of use is not limited to this, and kits for any of the above-mentioned purposes can be formed. Such a kit is also included in the scope of the present invention.
[0072]
【Example】
Example 1
FIG. 2 is an example of a nucleic acid probe-immobilized substrate according to an embodiment of the present invention. FIG. 2 is a so-called DNA chip for determining the type of hepatitis C virus (hereinafter referred to as HCV). In each of the solid-phased regions 2 of the nucleic acid probe-immobilized substrate A in FIG. 2A, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, a synthetic oligonucleotide probe consisting of the nucleic acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 was immobilized. Probes that respond to HCV type 1b are SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, probes that respond to HCV type 2a are SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, Probes that respond to HCV type 2b are SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 (FIG. 2A). Here, the probe consisting of SEQ ID NO: 5 reacts with both HCV 2a and 2b. Amino groups were introduced into the ends of these probes, spotted on polylysine-treated slide glass, and dried and fixed.
[0073]
As a target, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: with respect to the immobilized region 2 of the nucleic acid probe-immobilized substrate B shown in FIG. A nucleic acid probe consisting of the nucleotide sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 was immobilized (FIG. 2B).
[0074]
Example 2
HCV typing was performed using the nucleic acid probe-immobilized substrate prepared in Example 1. After amplifying the HCV genome extracted from the serum with the primers of SEQ ID NO: 21 and SEQ ID NO: 22, a re-PCR reaction was carried out using SEQ ID NO: 23 labeled with sequence biotin and the primer of SEQ ID NO: 22 labeled with cy5. Was. After the reaction, single-stranded DNA was obtained using avidin-modified magnetic fine particles (Magnetex-SA, Takara Shuzo). The Cy5-labeled target was dissolved in a 2 × SSC solution and reacted with the nucleic acid probe-immobilized substrates A and B shown in FIGS. 2A and 2B to determine the type of HCV. As a result, the optimal temperature was 40 ° C. for the conventional nucleic acid probe-immobilized substrate B, whereas the specificity was highest at 35 ° C. for the nucleic acid probe-immobilized substrate A according to the present invention. The fluorescence intensity of the nucleic acid probe-immobilized substrate A and that of the nucleic acid probe-immobilized substrate B were substantially the same.
[0075]
Example 3
FIG. 3 is a diagram showing a schematic configuration of a nucleic acid probe-immobilized substrate for electrochemical detection according to an embodiment of the present invention. In FIG. 3, the nucleic acid probe-immobilized base includes an electrode 12 disposed on a substrate 11 and a nucleic acid probe immobilized on the electrode 12. The immobilization of the nucleic acid probe on the electrode was carried out by introducing a thiol group into the end of each nucleic acid probe and immobilizing it on a patterned gold electrode.
[0076]
The nucleic acid probe used in this example is a nucleic acid probe for determining the type of hepatitis C virus (HCV). In each of the electrodes of the nucleic acid probe-immobilized substrate C shown in FIG. 2, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, the synthetic oligonucleotide probe represented by the nucleotide sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10 was immobilized (FIG. 3C). Probes that respond to HCV type 1b are SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, probes that respond to HCV type 2a are SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, Probes that respond to HCV type 2b are SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 10. Here, the probe 5 consisting of SEQ ID NO: 5 reacts with both 2a and 2b.
[0077]
As a target, the nucleic acid probe-immobilized substrate D has SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: Synthetic oligonucleotide probes consisting of 20 nucleic acid sequences were immobilized (FIG. 3D).
[0078]
Example 4
HCV typing was performed using the nucleic acid probe-immobilized substrates C and D prepared in Example 3 described above. After the HCV genome extracted from the serum was amplified with the primers of SEQ ID NO: 21 and SEQ ID NO: 22, a re-PCR reaction was performed with the primers of SEQ ID NO: 23 and SEQ ID NO: 22 labeled with sequence biotin. After the reaction, single-stranded DNA was obtained using avidin-modified magnetic fine particles (Magnetex-SA, Takara Shuzo). The single-stranded target was dissolved in a 2 × SSC solution, and a hybridization reaction was performed on the nucleic acid probe-immobilized substrate C and the nucleic acid probe-immobilized substrate D. Finally, the current of Hoechst 33258 was measured. As a result, the optimum temperature was 40 ° C. for the nucleic acid probe-immobilized substrate D, but the specificity was highest at 35 ° C. for the nucleic acid probe-immobilized substrate C. The current intensity of the nucleic acid probe-immobilized substrate C was about 1.2 times that of the nucleic acid probe-immobilized substrate D, and the background current was also reduced. From the above results, it was confirmed that the hybridization efficiency was improved and the background was reduced.
[0079]
As described above, the use of the nucleic acid probe of the present invention has improved the detection sensitivity as compared with the related art. This is due to the hairpin structure resulting from the binding of the nucleic acid probe to the target sequence, so that more intercalating agents are bound compared to conventional probes, resulting in increased current intensity and increased current signal. It is suggested that this was done.
[0080]
Example 5
After the HCV genome extracted from the serum was amplified with the primers of SEQ ID NO: 21 and SEQ ID NO: 22, a re-PCR reaction was performed with the primers of SEQ ID NO: 23 and SEQ ID NO: 22. After amplification with the primers of SEQ ID NOS: 24 and 25, a re-PCR reaction was performed with the primers of SEQ ID NOs: 26 and 25. The annealing temperature of the PCR reaction was 55 ° C and 60 ° C. As a result of analyzing these PCR products by electrophoresis, in the case of SEQ ID NO: 21 and SEQ ID NO: 22, non-specific amplification was observed at 55 ° C., and no non-specific signal was observed at 60 ° C. Was few. On the other hand, when the primers of SEQ ID NOs: 24 and 25 were used, it was found that the primers were specific even at 55 ° C. and had high amplification efficiency. As described above, the use of the primer according to the present invention enables a PCR reaction to be performed specifically and efficiently even at a low temperature.
[0081]
Accordingly, a means has been provided which enables simple and highly sensitive detection and / or extension of a nucleic acid chain. In addition, such various means according to the present invention can achieve desired detection and / or elongation at low cost, in addition to simple and high sensitivity.
[0082]
【The invention's effect】
According to the present invention, a nucleic acid fragment that specifically binds to a target sequence, a nucleic acid probe for specifically and highly sensitively detecting the target sequence, a primer that specifically and efficiently hybridizes to the target sequence, And a kit comprising them.
[0083]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of an example of a nucleic acid fragment according to an embodiment of the present invention.
FIG. 2 is a plan view showing a schematic configuration of a nucleic acid probe-immobilized substrate according to Examples 1 and 2.
FIG. 3 is a plan view showing a schematic configuration of a nucleic acid probe-immobilized substrate according to Examples 3 and 4.
FIG. 4 is a graph showing the results of a comparative experiment according to Example 4.
[Explanation of symbols]
1. Substrate 2. Immobilization area 11. Substrate 12. Electrode 13. Pat

Claims (9)

A nucleic acid fragment for hybridizing to a target nucleic acid comprising a first target sequence x1 and a second target sequence x2, wherein the sequence X1 is complementary to the target sequence x1, an arbitrary sequence Y, an arbitrary sequence Z, A nucleic acid fragment comprising a sequence y complementary to the sequence Y and a sequence X2 complementary to the target sequence x2, and represented by X1-YZ-y-X2. A nucleic acid fragment for hybridizing to a target nucleic acid containing a sequence from the first target sequence x1 to the (n + 1) th target sequence xn + 1, wherein the sequence X1, the arbitrary sequence Y1, the arbitrary sequence complementary to the target sequence x1 From X1-Y1-Z1-y1 consisting of Z1 and the sequence y1 complementary to the sequence Y1, a sequence Xn complementary to the target sequence xn, an arbitrary sequence Yn, an arbitrary sequence Zn and a sequence complementary to the sequence Yn yn and Xn-Yn-Zn-yn and a sequence Xn + 1 complementary to the target sequence xn + 1, and X1-Y1-Z1-y1... -Xn-Yn-Zn-yn-Xn + 1. (Where n is an integer of 2 or more). The nucleic acid fragment according to claim 1, wherein the complementarity between the sequences Y1 and y1 is 90% or more. The nucleic acid fragment according to any one of claims 1 to 3, wherein the length of each of the sequence X1 and the sequence X2 is 5 to 9 bases. The primer according to any one of claims 1 to 4, which is used as a reagent selected from the group consisting of a primer for extending the target nucleic acid and a nucleic acid probe for detecting the target nucleic acid. Nucleic acid fragments. A nucleic acid probe-immobilized substrate comprising the nucleic acid fragment according to any one of claims 1 to 4 as a nucleic acid probe. An assay kit comprising the nucleic acid fragment according to any one of claims 1 to 4 as a reagent selected from the group consisting of a primer and a nucleic acid probe. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 A nucleic acid probe for detecting human hepatitis C virus represented by the base sequence described above. A primer for amplifying a nucleic acid derived from human hepatitis C virus represented by a base sequence described in a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

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