JP2004073065A - 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 x and a second target sequence x' and comprises sequence X complementary to the sequence x, a spacer S and a sequence X' complementary to the sequence x' and is represented by X-S-X'. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】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２】実施例１に係る核酸プローブの１例を示す図。
【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５の実施形態に係るプライマーの１例を示す図。
【図５】実施例４に係る検出結果の１例を示す図。
【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. A method of confirming or detecting an amplification product using a substrate or the like on which a complementary nucleic acid probe is immobilized is generally used. In that case, in order to specifically detect a specific nucleic acid strand, a nucleic acid 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]
Further, a current detection type DNA chip using a nucleic acid probe-immobilized electrode and an intercalating agent active in an electrochemical hand has been reported. This method has advantages such as low running cost since no label is required, and no need for an expensive and large detection device as in the case of fluorescence detection. It is generally considered that a longer nucleic acid probe has higher detection sensitivity, but since the intercalating agent slightly reacts with the nucleic acid probe, the use of a longer nucleic acid probe increases noise, resulting in insufficient detection sensitivity. There is such a problem.
[0004]
Furthermore, when performing a specific reaction using a long nucleic acid probe, the melting temperature (Tm) becomes high. Therefore, a harmful additive such as dimethylsulfoxide (DMSO) or dimethylformamide is added to lower the temperature. I needed 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 present invention provides the following means for solving the above problems. That is,
A nucleic acid fragment for hybridizing to a target nucleic acid containing a first target sequence x and a second target sequence x ′, comprising a sequence X complementary to sequence x, a spacer S, and a sequence complementary to sequence x ′. A nucleic acid fragment comprising a specific sequence X ′ and represented by XSX ′;
A nucleic acid fragment for hybridizing to a target nucleic acid including a sequence from the first target sequence x1 to the (n + 1) th target sequence xn + 1, wherein the sequence X1 is complementary to the target sequence x1, a first spacer S1, It includes a sequence Xn complementary to the target sequence xn, an n-th spacer Sn, and a sequence Xn + 1 complementary to the sequence xn + 1 and is represented by X1-S1 -...- Xn-Sn-Xn + 1. (Where n is an integer greater than or equal to 2);
A nucleic acid probe-immobilized substrate comprising any one of the above nucleic acid fragments as a nucleic acid probe;
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 NOs: 1 and 11; SEQ ID NOs: 2 and 12; SEQ ID NOs: 3 and 13; SEQ ID NOs: 4 and 14; SEQ ID NOs: 5 and 15; 6 and SEQ ID NO: 16, SEQ ID NO: 7 and SEQ ID NO: 17, SEQ ID NO: 8 and SEQ ID NO: 18, SEQ ID NO: 9 and SEQ ID NO: 19, and SEQ ID NO: 10 and SEQ ID NO: 20 A nucleic acid probe for detecting human hepatitis C virus containing the above-described nucleic acid fragment; and the sequences X and X 'are those having SEQ ID NOs: 34 and 37, SEQ ID NOs: 35 and 38, and SEQ ID NOs: 36 and 36 A primer for extending human hepatitis C virus, comprising the nucleic acid fragment described above, which is selected from the group consisting of No. 39;
It is.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
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.
[0008]
The target nucleic acid to which the nucleic acid fragment according to the embodiment of the present invention hybridizes includes a target sequence x and a second target sequence x ′. At this time, the first target sequence and the second target sequence may be adjacent to each other and may be continuous sequences on the target nucleic acid including both sequences, or may be sequences that are located at distant positions in the target nucleic acid. There may be.
[0009]
On the other hand, the nucleic acid fragment according to the embodiment of the present invention includes a sequence X complementary to the target sequence x and a sequence X ′ complementary to the target sequence x ′, and includes a spacer S therebetween. That is, the nucleic acid fragment is a sequence represented by the sequence represented by the general formula “XSX ′”.
[0010]
Here, the sequence “x” and the sequence “x ′” (here, x is lowercase) included in the target sequence, and the sequence “X” and the sequence “X ′” (where X is uppercase) contained in the nucleic acid fragment are included. Are complementary to each other. Further, in the present specification, for the sake of convenience, when the same alphabet is indicated by uppercase and lowercase letters, it indicates that they are sequences comprising base sequences complementary to each other.
[0011]
When the nucleic acid fragment according to the invention hybridizes with the target sequence, the spacer is present without binding to the target sequence. Further, when the nucleic acid fragment and the target sequence are hybridized, the spacer may form a bond such as hybridization within its own sequence if desired, or may form any bond as desired. It may exist without being done.
[0012]
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.
[0013]
The term "nucleic acid" as used herein generally refers to any structure thereof, including any nucleic acid analogs such as DNA and RNA, PNA (ie, peptide nucleic acids) and LNA (ie, locked nucleic acids). It is a general term for a substance that can be represented by a base sequence. Such a nucleic acid may be a naturally occurring one or an artificially synthesized one.
[0014]
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 y or mass spectroscopy.
[0015]
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.
[0016]
As used herein, the term "spacer" refers to a chain substance having a certain length arranged between sequences complementary to a target sequence among the sequences contained in the nucleic acid fragment according to the embodiment of the present invention. Say. Examples of the substance used as the spacer may be an organic chain molecule, such as a nucleic acid, a peptide, a polypeptide, an alkane chain, polyvinyl alcohol, and polyethylene glycol.
[0017]
The lengths of the sequence X and the sequence X 'are not particularly limited, but may be 5 to 100 bases, preferably 5 to 9 bases, respectively. Furthermore, the total length of the sequence X and the sequence X 'is not particularly limited, but may be 10 to 200 bases, preferably 10 to 18 bases.
[0018]
The length of the spacer may be from about 6 to about 1000, preferably from about 20 to about 200. Alternatively, the number of atoms constituting the spacer may be 3 or more and 10 or less, 10 or more and 100 or less, or 100 or more and 500 or less. In the case of the respective lengths, electrons can move efficiently when the length is 10 atoms or less. Therefore, there is an advantage in the electrochemical nucleic acid detection method. On the other hand, when the number is 100 atoms or more, the problem of steric hindrance of the substrate can be solved, so that the efficiency of the hybridization reaction can be increased. When it is 10 atoms or more and 100 atoms or less, both characteristics can be provided, and there is an advantage that high detection sensitivity can be achieved in an electrochemical nucleic acid chain detection method. In the case of nucleic acids, the spacer may be about 1 to about 200 bases, preferably about 3 to about 50 bases, and more preferably about 5 to about 30 bases. The longer the length of the nucleic acid chain, the higher the efficiency of hybridization. However, the synthesis of a long synthetic nucleic acid is difficult and the yield is poor. Further, there is a problem that the background is increased by the electrochemical detection method. On the other hand, a short nucleic acid chain has low reaction efficiency, but does not easily cause a background problem. Therefore, about 5 to 30 bases can be said to be the optimal length.
[0019]
Further, the nucleic acid fragment according to the present invention has a spacer S that is not involved in hybridization with the target sequence. In the above-described example, an example in which the spacer in the nucleic acid fragment is included by 1 has been described. However, the present invention is not limited thereto, and a plurality of spacers may be included in one nucleic acid fragment. In this case, it is preferable that a sequence complementary to the target sequence is linked to both ends of each spacer. When a plurality of spacers are included, the spacers may be equal, all may be different, or may be partially equal.
[0020]
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, the nucleic acid fragment comprises a sequence X1 complementary to the first target sequence x1 and a spacer S1. A sequence X (n + 1) complementary to the (n + 1) th target sequence xn + 1 is further added to a continuous sequence from X1-S1 to Xn-Sn comprising the sequence Xn complementary to the nth target sequence xn and the spacer Sn. The nucleic acid fragment may include any nucleic acid fragment represented by X1-S1-... -Xn-Sn-Xn + 1. Here, n is an integer of 2 or more.
[0021]
When a plurality of spacers are included, the length of the nucleic acid fragment is not particularly limited, but may be about 50 to about 3000, preferably about 100 to about 500, including the spacer portion. The total length of the nucleic acid constituting the portion other than the spacer of the nucleic acid fragment is not particularly limited, but may be about 10 to about 500 bases, and preferably about 10 to about 50 bases. Just fine.
[0022]
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.
[0023]
Further, since the Tm can be set at a low temperature while maintaining the specificity, the hybridization reaction can be performed at a low temperature without adding a special additional reagent.
[0024]
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.
[0025]
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.
[0026]
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.
[0027]
2. Nucleic acid probe The nucleic acid probe according to the 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 or captured. 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.
[0028]
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.
[0029]
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. Alternatively, a chemical modification known per se may be performed. Such modified nucleic acid probes are also provided as the present invention.
[0030]
The total length of the nucleic acid probe according to the embodiment of the present invention, including the spacer portion, may be about 50 ° to about 3000 °, preferably about 100 ° to about 500 °. The total length of the nucleic acid constituting the portion other than the spacer of the nucleic acid fragment is not particularly limited, but may be about 10 to about 500 bases, and preferably about 10 to about 50 bases. Just fine. Further, even if a part thereof contains a substance that is not a nucleic acid, for example, such a substance as a spacer, it is called a nucleic acid probe.
[0031]
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.
[0032]
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.
[0033]
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. Also, microbeads, microtiter plates, and the like can be used as a substrate for immobilizing a nucleic acid probe.
[0034]
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.
[0035]
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.
[0036]
The nucleic acid probe-immobilized substrate used in the embodiment of the present invention will be described below using examples.
[0037]
(1) First Example FIG. 1 schematically shows a first example of a nucleic acid probe-immobilized substrate that can be used according to an 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. 1).
[0038]
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.
[0039]
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.
[0040]
(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.
[0041]
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. .
[0042]
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.
[0043]
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.
[0044]
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, Examples include a noble metal electrode such as rhodium, an oxide electrode such as titanium oxide, tin oxide, manganese oxide, and lead oxide; a semiconductor electrode 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 another surface treating agent as desired.
[0045]
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 to the substrate or the electrode via an additional 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.
[0046]
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.
[0047]
(3) Detection The nucleic acid probe-immobilized substrate according to the present invention is used 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. It is possible to use electrochemical methods and fluorescence detection methods.
[0048]
(A) Electrochemical detection The double-stranded nucleic acid can be detected electrochemically using, for example, a double-stranded recognition substance known per se.
[0049]
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.
[0050]
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.
[0051]
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.
[0052]
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.
[0053]
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.
[0054]
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.
[0055]
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.
[0056]
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.
[0057]
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.
[0058]
Further, since the Tm can be set at a low temperature while maintaining the specificity, the hybridization reaction can be performed at a low temperature without adding a special additional reagent. It is possible to perform a PCR reaction.
[0059]
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.
[0060]
(B) Fluorescence detection method In the case of a method using a fluorescent labeling substance, the antisense primer is labeled with a substance capable of obtaining a fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Is also good. 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.
[0061]
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.
[0062]
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.
[0063]
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.
[0064]
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.
[0065]
When the probe according to the embodiment of the present invention is used, it is possible to greatly improve the S / N in electrochemical nucleic acid detection utilizing an electrochemical reaction of an intercalating agent. The cause of noise in electrochemical nucleic acid detection is a signal derived from an intercalating agent that binds to a single-stranded probe portion. In addition, the binding amount of the intercalating agent increases as the length of the nucleic acid probe increases. Therefore, a short nucleic acid probe is preferable to reduce noise. However, there is a problem that a short nucleic acid probe of 9 bases or less does not provide sufficient specificity. In the present invention, the overall length is increased by linking a short nucleic acid probe to a spacer, thereby successfully reducing noise without sacrificing specificity.
[0066]
4. Primers 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.
[0067]
The total length of a primer according to an embodiment of the present invention, including the spacer portion, may be from about 50 to about 3000, preferably from about 100 to about 500. The total length of the nucleic acid constituting the portion other than the spacer of the nucleic acid fragment is not particularly limited, but may be about 10 to about 500 bases, and preferably about 10 to about 50 bases. Just fine.
[0068]
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.
[0069]
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.
[0070]
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
[0071]
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.
[0072]
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, hybridization and extension can be achieved at a low temperature without adding a special additional reagent.
[0073]
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.
[0074]
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 provided as the present invention by forming a kit in combination with a substrate and / or various reagents and / or labeling substances. May be done. 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.
[0075]
【Example】
Example 1
FIG. 1 is an example of a nucleic acid probe-immobilized substrate according to an embodiment of the present invention. FIG. 1 is a so-called DNA chip for determining the type of hepatitis C virus (HCV). In each of the immobilized regions 2 of the nucleic acid probe-immobilized substrate A shown in FIG. 1, 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, and 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: as sequence X ′ 17, a nucleic acid probe comprising a ligated synthetic oligonucleotide comprising SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, and two hexaethylene glycol units as spacers was immobilized. The configuration of each nucleic acid probe is as shown in FIG. That is, ten types of probes from nucleic acid probe 1 to nucleic acid probe 10 shown in FIG. 2 were immobilized on each immobilization region for each type. The respective combinations are as follows: SEQ ID NO: 1 and SEQ ID NO: 11, SEQ ID NO: 2 and SEQ ID NO: 12, SEQ ID NO: 3 and SEQ ID NO: 13, SEQ ID NO: 4 and SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 15, SEQ ID NO: 6 and SEQ ID NO: No. 16, SEQ ID NO: 7 and SEQ ID NO: 17, SEQ ID NO: 8 and SEQ ID NO: 18, SEQ ID NO: 9 and SEQ ID NO: 19, and SEQ ID NO: 10 and SEQ ID NO: 20.
[0076]
The nucleic acid probes that react with HCV type 1b are nucleic acid probe 1, nucleic acid probe 4, and nucleic acid probe 6 shown in FIG. 2, and the nucleic acid probes that react with HCV type 2a are nucleic acid probe 2, nucleic acid probe 5, and nucleic acid probe 5 in FIG. The nucleic acid probes 7 and 9 that react with HCV type 2b are the nucleic acid probe 3, the nucleic acid probe 5, the nucleic acid probe 8 and the nucleic acid probe 10 in FIG. Here, the nucleic acid probe 5 reacts to both HCV 2a and 2b.
[0077]
The immobilization of the nucleic acid probes was carried out by introducing an amino group into the end of these nucleic acid probes, spotting them on a polylysine-treated slide glass, and drying and fixing them.
[0078]
As a target, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, sequence Nucleic acid probes consisting of the nucleotide sequences of Nos. 28, 29 and 30 were similarly immobilized (FIG. 2B).
[0079]
Example 2
HCV typing was performed using the nucleic acid probe-immobilized substrate prepared in Example 1 described above. After the HCV genome extracted from the serum was amplified with the nucleic acid primers of SEQ ID NO: 31 and SEQ ID NO: 32, a re-PCR reaction was performed with the nucleic acid primers of SEQ ID NO: 33 labeled with sequence biotin and SEQ ID NO: 32 labeled with cy5. 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 allowed to react with the nucleic acid probe-immobilized substrate A and the nucleic acid probe-immobilized substrate B to determine the type of HCV. As a result, the optimum temperature was 45 ° C. for the nucleic acid probe-immobilized substrate B, but the specificity was highest at 38 ° C. for the nucleic acid probe-immobilized substrate B. It was found that the fluorescence intensity of the nucleic acid probe-immobilized substrate A was almost the same as that of the nucleic acid probe-immobilized substrate B.
[0080]
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. FIG. 3 shows a nucleic acid probe-immobilized substrate including 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.
[0081]
In each of the immobilized regions 2 of the nucleic acid probe-immobilized substrate A shown in FIG. 3, for each region, 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, and 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: as sequence X ′ 17, a nucleic acid probe comprising a linked synthetic oligonucleotide comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and two hexaethylene glycol units as a spacer was immobilized. The configuration of each nucleic acid probe is as shown in FIG. That is, ten types of probes from nucleic acid probe 1 to nucleic acid probe 10 shown in FIG. 2 were immobilized on each immobilization region for each type.
[0082]
The nucleic acid probes that react with HCV type 1b are nucleic acid probe 1, nucleic acid probe 4, and nucleic acid probe 6 shown in FIG. 2, and the nucleic acid probes that react with HCV type 2a are nucleic acid probe 2, nucleic acid probe 5, and nucleic acid probe 5 in FIG. The nucleic acid probe 7 and the nucleic acid probe 9 which react with the HCV type 2b are the nucleic acid probe 3, the nucleic acid probe 5, the nucleic acid probe 8 and the nucleic acid probe 10 in FIG. Here, the nucleic acid probe 5 reacts to both HCV 2a and 2b.
[0083]
For the immobilized region 2 of the nucleic acid probe-immobilized substrate B shown in FIG. 3B as a target, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, sequence Nucleic acid probes consisting of the nucleotide sequences of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 were similarly immobilized (FIG. 3B).
[0084]
Example 4
HCV typing was performed using the chip manufactured in the third embodiment. After the HCV genome extracted from the serum was amplified with the primers of SEQ ID NO: 31 and SEQ ID NO: 32, a re-PCR reaction was performed with the primers of SEQ ID NO: 33 and SEQ ID NO: 32 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 A and the nucleic acid probe-immobilized substrate B. Finally, the current of Hoechst 33258 was measured. As a result, the optimal temperature was 35 ° C. for the nucleic acid probe-immobilized substrate B, but the specificity was highest at 40 ° C. for the nucleic acid probe-immobilized substrate A. The current intensity of the nucleic acid probe-immobilized substrate A was about 1.2 times that of the nucleic acid probe-immobilized substrate B, 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 (FIG. 5).
[0085]
As described above, by using the nucleic acid probe of the present invention, sufficient specificity can be obtained even when the length of a sequence directly involved in hybridization is short. In addition, the background can be reduced, that is, noise can be reduced.
[0086]
Example 5
After the HCV genome extracted from the serum was amplified with the primers of SEQ ID NO: 31 and SEQ ID NO: 32, a re-PCR reaction was performed with the primers of SEQ ID NO: 33 and SEQ ID NO: 32. Also, after amplification with the connecting primer 34 of SEQ ID NO: 34 and SEQ ID NO: 37 and the connecting primer 35 of SEQ ID NO: 35 and SEQ ID NO: 38, the connecting primer 36 of SEQ ID NO: 36 and SEQ ID NO: 39, and Re-PCR reaction was carried out using the ligation primer. The above ligated primer further contains two hexaethylene glycol units as a spacer. That is, the ligated primers used are as listed in FIG. 4 (FIG. 4). 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: 31 and SEQ ID NO: 32, non-specific amplification was observed at 55 ° C., and no non-specific signal was observed at 60 ° C. There were few. On the other hand, it was found that the use of the ligated primers of SEQ ID NO: 34 and SEQ ID NO: 35 was specific even at 55 ° C. and the amplification efficiency was high. From the above results, it was revealed that the PCR reaction can be performed specifically and with high efficiency by using the primer according to the present invention. The primers according to the invention have also made it possible to achieve hybridization and extension at low temperatures.
[0087]
【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 for specifically and efficiently hybridizing and extending to the target sequence, and Kits comprising them were provided.
[0088]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a plan view showing a schematic configuration of a nucleic acid probe-immobilized substrate according to Examples 1 and 2.
FIG. 2 is a diagram showing an example of a nucleic acid probe according to Example 1.
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 view showing one example of a primer according to the embodiment of Example 5.
FIG. 5 is a diagram illustrating an example of a detection result according to the fourth embodiment.
[Explanation of symbols]
1. Substrate 2. Immobilization area 11. Substrate 12. Electrode 13. Pat

Claims (10)

A nucleic acid fragment for hybridizing to a target nucleic acid containing a first target sequence x and a second target sequence x ′, comprising a sequence X complementary to sequence x, a spacer S, and a sequence complementary to sequence x ′. A nucleic acid fragment comprising a specific sequence X ′ and represented by XSX ′. 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 is complementary to the target sequence x1, a first spacer S1, It includes a sequence Xn complementary to the target sequence xn, an n-th spacer Sn, and a sequence Xn + 1 complementary to the sequence xn + 1 and is represented by X1-S1 -...- Xn-Sn-Xn + 1. (Where n is an integer of 2 or more). The nucleic acid fragment according to claim 1, wherein each of X and X 'has a length of 5 to 20 bases. The nucleic acid fragment according to any one of claims 1 to 3, wherein the spacer is selected from the group consisting of a nucleic acid, a polypeptide, an alkane, and polyethylene glycol. The nucleic acid fragment according to any one of claims 1 to 4, wherein the spacer has 3 to 500 atoms. The primer according to any one of claims 1 to 5, 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 5 as a nucleic acid probe. An assay kit comprising the nucleic acid fragment according to any one of claims 1 to 5 as a reagent selected from the group consisting of a primer and a nucleic acid probe. SEQ ID NOS: 1 and 11; SEQ ID NOs: 2 and 12; SEQ ID NOs: 3 and 13; SEQ ID NOs: 4 and 14; SEQ ID NOs: 5 and 15; 6 and SEQ ID NO: 16, SEQ ID NO: 7 and SEQ ID NO: 17, SEQ ID NO: 8 and SEQ ID NO: 18, SEQ ID NO: 9 and SEQ ID NO: 19, and SEQ ID NO: 10 and SEQ ID NO: 20 A nucleic acid probe for detecting a human hepatitis C virus containing the nucleic acid fragment according to claim 1. The sequence of claim 1, wherein the sequences X and X 'are selected from the group consisting of SEQ ID NOs: 34 and 37, SEQ ID NOs: 35 and 38, and SEQ ID NOs: 36 and 39. A primer for extending human hepatitis C virus containing a nucleic acid fragment.

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