JP2014176297A - Base-supporting labeling method, base sequence information acquisition method, and base-supporting labeled single-stranded nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that enables bases in nucleic acid to be indirectly labeled at a high labeling rate, while preserving base sequence information of the nucleic acid.SOLUTION: There is provided a base-supporting labeling method that involves indirectly labeling the bases in a single-stranded nucleic acid while preserving base sequence information the nucleic acid has, wherein the method includes a procedure for inserting an oligonucleotide L, which has been labeled in a specific and highly-precise manner according to the type of base constituting a nucleotide, in the 3'-terminal or 5'-terminal of the nucleotide. According to the base-supporting labeling method, each base in the single-stranded nucleic acid can be identified with high precision according to the inserted oligonucleotide L. Therefore, by identifying the oligonucleotide L label under an electron microscope, for example, highly-precise base sequence information of the single-stranded nucleic acid can be acquired.

Description

  The present invention relates to a base-corresponding labeling method, a base sequence information acquisition method, and a base-corresponding labeled single-stranded nucleic acid. More specifically, the present invention relates to a base-corresponding labeling method for performing indirect labeling suitable for identification under an electron microscope on a base in a nucleic acid.

  There is a need for a technique for easily and rapidly acquiring base sequence information of oligonucleotides such as DNA and RNA, or polynucleotides (nucleic acids). In particular, genomic drug discovery that identifies genes that cause diseases and constitutions by analyzing genetic information collected from a large number of patients and / or healthy individuals, and that targets those gene products (proteins) In order to realize and advance customized medicine that seeks to provide optimal medical care according to genetic information, it is necessary to further reduce the cost and speed of the technology.

  As a base sequence information acquisition technique of nucleic acid, each base of adenine (A), thymine (T), guanine (G), cytosine (C), uracil (U) is utilized by utilizing the resolution and high resolution of an electron microscope. Techniques for identifying are being developed. In this technique, each base is directly labeled with a different heavy atom or heavy atom cluster, and the base is identified based on the labeled heavy atom under an electron microscope.

  Patent Document 1 discloses a base modification method that enables base identification under an electron microscope. According to this base modification method, the base in the nucleic acid can be directly labeled for each base species by chemical means while maintaining the base sequence information of the nucleic acid.

  In relation to the present invention, Patent Documents 2 and 3 disclose techniques capable of reading sequence information at high speed. This technology is a next-generation base sequence determination technology (sequencing technology) called “Optipore”, which is currently being developed, and specifically includes the following procedures.

  First, in a circular DNA conversion (Circular DNA Conversion (CDC)) method (see Patent Document 4), a single-stranded nucleic acid to be sequence-read (hereinafter described as DNA and referred to as target ssDNA) An oligonucleotide having a predetermined number of bases is inserted and added to the 3 ′ end or 5 ′ end of the nucleotide. This oligomer is specifically designed for each base of A, G, C, and T, and is inserted into the target ssDNA while retaining the base sequence information of the nucleotide at the insertion site.

  Next, a single-stranded DNA (beacon) having a base sequence complementary to the oligonucleotide and labeled with a fluorescent reagent for base discrimination is bound (hybridized) to the target ssDNA to which the oligonucleotide is inserted and added, Double stranded DNA is formed.

  Then, the formed double-stranded DNA is passed through the solid state nanopore. When the double-stranded DNA passes through the nanopore, a beacon is released from the target ssDNA, and the fluorescent reagent that has been quenched on the beacon emits light. By detecting this fluorescence with a microscope-CCD camera, the base sequence of the target ssDNA is read.

International Publication No. 2009/020249 International Publication No. 2006/020775 Special table 2008-509671 International Publication No. 2010/053820

  In order to apply the above-mentioned technology for obtaining nucleic acid base sequence information using an electron microscope as a sequencing technology, the base in the target nucleic acid is labeled at a sufficiently high rate in order to improve sequence reading accuracy. A base labeling method that makes all bases distinguishable is essential.

  Therefore, the main object of the present invention is to provide a technique for indirectly labeling a base in a nucleic acid at a high labeling rate while maintaining the base sequence information of the nucleic acid.

In order to solve the above-mentioned problems, the present invention is a method for labeling a base in a single-stranded nucleic acid while maintaining the base sequence information of the nucleic acid. Provided is a base-corresponding labeling method including a procedure for inserting an oligonucleotide that is specifically and highly labeled according to the type of base constituting the nucleotide on the end side or 5 ′ end side.
According to this base-corresponding labeling method, each base in a single-stranded nucleic acid can be reliably labeled with the inserted oligonucleotide. Therefore, for example, by identifying the label of the oligonucleotide under an electron microscope, Nucleotide base sequence information can be obtained with high accuracy.
In this base-corresponding labeling method, the oligonucleotide can be inserted by a circular DNA conversion method.
In this base-corresponding labeling method, the oligonucleotide is an oligonucleotide labeled with a metal cluster having an average particle diameter of 1-1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5-1000 nanometers. Can be used. It is preferable to use four types of metal clusters having different average particle diameters. Moreover, it is preferable to use 4 types from which an average particle diameter and / or light emission wavelength differ as said inorganic fluorescent substance particle.
In this base-corresponding labeling method, it is preferable to use the inorganic phosphor particles and / or oligonucleotides separated and purified after labeling the inorganic phosphor particles as the oligonucleotides.

The present invention also provides a single-stranded nucleic acid in which an oligonucleotide labeled with a specific label according to the type of the base is inserted at the 3 ′ end or 5 ′ end of the nucleotide containing the base to be read. To do.
In this single-stranded nucleic acid, the label can be a metal cluster having an average particle diameter of 1 to 1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5 to 1000 nanometers.

  According to the present invention, there is provided a technique for labeling and identifying a base in a nucleic acid at a high labeling rate while maintaining the base sequence information of the nucleic acid.

It is a figure explaining the structure of the double strand for conversion. It is a figure explaining the labeled oligomer labeled with undecagold and nanogold. It is a figure explaining the target ssDNA by which the adapter arrangement | sequence was added to 5 'terminal and was solid-phased on the solid-phase surface. It is a figure explaining the hybridization reaction of target ssDNA and the double strand for conversion. It is a figure explaining circularization reaction of the composite_body | complex of target ssDNA and the double strand for conversion. It is a figure explaining the truncation reaction of the nucleotide from 3 'end of target ssDNA. It is a figure explaining the dissociation reaction of the double strand for conversion. It is a figure explaining the structure of conversion ssDNA.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  In the base-corresponding labeling method according to the present invention, specific and high-precision labeling is performed in advance on the 3 ′ end side or 5 ′ end side of the nucleotide in the single-stranded nucleic acid according to the type of base constituting the nucleotide. By inserting an oligonucleotide (hereinafter referred to as “labeled oligomer”), the base is labeled while maintaining the base sequence information of the nucleic acid.

  Insertion of a labeled oligomer can be performed using a circular DNA conversion method. The CDC method is a method in which different types of oligonucleotides can be inserted for each type of base of nucleotides at the 3 ′ end or 5 ′ end of nucleotides in a single-stranded nucleic acid, and there are several variations. . Hereinafter, a method for obtaining base sequence information of a single-stranded nucleic acid by indirectly labeling a base in the single-stranded nucleic acid in a base-corresponding manner by inserting a labeled oligomer according to the procedure of the example will be described.

  However, in the base-corresponding labeling method according to the present invention, it is possible to adopt an insertion method other than the CDC method as long as the target labeled oligomer can be inserted. The procedure exemplified below (Patent Document 4) Naturally, it is possible to adopt a variation of the CDC method different from that of the reference).

1. Construction of double-stranded library for conversion [Conversion double-stranded structure]
A plurality of “double strands for conversion” that function to insert labeled oligomers into a single-stranded nucleic acid (target ssDNA) to be sequence-readed are prepared and constructed as a library. The conversion duplex contains a labeled oligomer in one of the duplexes. FIG. 1 shows the structure of the double strand for conversion. The conversion duplex has a double-stranded portion, a first overhang and a second overhang.

One of the double-stranded parts is constituted by a labeled oligomer L. The labeled oligomer L has a base sequence Xx and a base sequence R that serves as a recognition site for a type II restriction enzyme. The labeled oligomer has a specific label depending on the type of base (x) to be identified in the target ssDNA (corresponding to x ₁ , x ₂ , x ₃ , x ₄ , x ₅ , x ₆ in FIG. 4). Has been. The symbol x ′ shown in the second overhang indicates a base complementary to this labeled base (x).

The base sequence Xx includes a known base sequence (5′-S ₁ -S ₂ -S ₃ -S ₄ -S _{5 -3 ′} ) at the _{5 ′ end} . This base sequence (hereinafter referred to as “adapter sequence”) is the same as the sequence added to the 5 ′ end of the target ssDNA in the procedure described later.

  The other of the double strands has a base sequence X′x and a base sequence R ′ that are complementary to the base sequence Xx and the base sequence R, respectively, and further the first on the 5 ′ end side of the base sequence R ′. The second overhang is located on the 3 ′ end side of the base sequence X′x.

The first overhang includes a nucleotide having at least one random base (n) at a position adjacent to the 5 ′ end of the nucleotide of the base sequence R ′ (n is 1 in the figure). The base (n) is any one of A, G, T, and C. Furthermore, the first overhang is a base sequence complementary to the adapter sequence (5′-S ₁ -S ₂ -S ₃ -S ₄ -S _{5 -3 ′} ) added to the 5 ′ end of the target ssDNA. having _{(5'-S 5 '-S} 4'-S 3 '-S 2'-S 1 '-3').

  The second overhang includes a nucleotide having a base (x ′) complementary to the base (x) to be identified in the target ssDNA at a position adjacent to the 3 ′ end of the nucleotide of the base sequence X′x. The base (x ′) is any one of A, G, T, and C. Furthermore, the second overhang contains nucleotides with at least 3 random bases (n) (n is 5 in the figure).

  The base length of the first overhang and the second overhang is 3 to 12 mer. The base length of the base sequence Xx of the labeled oligomer L (including the adapter sequence) is appropriately set in the range of 4 to 25 mer depending on the type of type II restriction enzyme used. Of the base sequence Xx, the base sequence other than the adapter sequence and the length thereof are not particularly limited as long as the label described later is possible. The base sequence of the base sequence R of the restriction enzyme recognition site of the labeled oligomer L and its length can be appropriately designed according to the type of type II restriction enzyme used. The length of the base sequence Xx is such that the enzyme cleavage site is 3 ′ of the target ssDNA in the procedure of treating the complex of the circularized target ssDNA and the double strand for conversion with a type II restriction enzyme (described in detail later). The length is designed so that at least one nucleotide can be cut off from the terminal.

  The size of the library depends on the number of nucleotides with random base n. For example, in the example where n is 6 shown in the figure, the conversion duplex contained in the library is the seventh power of 4 (4 types of A, G, T, and C) (the number of bases n 6 and the base x The total number of '1') is 16,384.

[Labeling of labeled oligomer]
The label of the labeled oligomer L is specific depending on the type of the base x ′ located in the second overhang (that is, the type of the base x to be labeled in the target ssDNA complementary thereto). .

  The label of the labeled oligomer L is obtained by chemically treating the above-described oligonucleotide having a base length with a metal cluster having an average particle diameter of 1 to 1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5 to 1000 nanometers. This can be done by bonding to The label oligomer L can be labeled with one or both of the metal cluster and the inorganic phosphor particles.

The labeling with metal clusters is preferably performed by assigning four types of metal clusters having different average particle diameters to the respective labeled oligomers for labeling A, G, T, and C. An example of the label is shown below. According to this labeling, it is possible to identify the type of base corresponding to the labeled oligomer by reading the particle size of the metal cluster labeled on the labeled oligomer under an electron microscope.
Labeled oligomers for labeling nucleotide A _{L A:} labeled oligomers for metal clusters labeled nucleotide G of a particle diameter 5 nm _{L G:} labeled oligomers for metal clusters labeled nucleotide T of grain size 10 nm _{L T:} particle size 15nm of metal clusters labeled bases Labeled oligomer L C for _C : metal cluster with a particle size of 20 nm

Alternatively, labeling with metal clusters can also be performed by labeling two types of metal clusters having different average particle diameters to the respective label oligomers for A, G, T, and C in different combinations. For example, when using two kinds of gold colloids of undecagold (gold 11 atoms) and nanogold (gold 50 atoms) as the metal cluster, for example, labeling is performed as follows (see also FIG. 2). Also in this case, it is possible to identify the type of base corresponding to the labeled oligomer by reading the particle diameter of the gold colloid labeled on the labeled oligomer under an electron microscope.
Labeled oligomer L for labeled base A _A : Undecagold + Undecagold Labeled oligomer L for labeled base G _G : Undecagold + Nanogold Labeled oligomer L for labeled base T _T : Nanogold + Undecagold Label base C for the labeled oligomer _{L C:} nanogold + nanogold

  The labeling with inorganic phosphor particles can also be performed using inorganic phosphor particles having different emission wavelengths. Examples of the inorganic phosphor particles include particles made of rare earth element-activated metal oxides, metal sulfides or metal sulfates, halophosphate compounds, and the like having emission wavelengths in the red, green, or blue regions. The following particles made of phosphors can be used. As the inorganic phosphor particles, those obtained by coating semiconductor atoms (cadmium mixed with selenium or tellurium) with a semiconductor shell (zinc sulfide), which is commercially available under the registered trademark “Qdot”, can also be used.

red:
Y ₂ O ₂ S: Eu ³⁺
Gd ₂ O ₂ S: Eu ³⁺
YVO ₄ : Eu ³⁺
Y ₂ O ₂ S: Eu, Sm
SrTiO ₃ : Pr
BaSi ₂ Al ₂ O ₈ : Eu ²⁺
BaMg ₂ Al ₁₆ O ₂₇ : Eu ²⁺
Y _0.65 Gd _0.35 BO ₃ : Eu ³⁺
La ₂ O ₂ S: Eu ³⁺ , Sm

green:
Ba ₂ SiO ₄ : Eu ²⁺
Zn (Ga, Al) ₂ O ₄ : Mn
Y ₃ (Al, Ga) ₅ O ₁₂ : Tb
Y ₂ SiO ₅ : Tb
ZnS: Cu,
Zn ₂ SiO ₄ : Mn

Blue:
BaAl ₂ Si ₂ O ₈ : Eu ²⁺
BaMgAl ₁₄ O ₂₃ : Eu ²⁺
Y ₂ SiO ₅ : Ce
ZnGa ₂ O ₄
ZnS: Ag, Cl

For example, the labeling is performed as follows. According to this label, it is possible to identify the type of the base labeled by the labeled oligomer by reading the cathodoluminescence emission wavelength of the inorganic phosphor particles labeled with the labeled oligomer under an electron microscope. If phosphor particles having different particle diameters are used, the particle diameter can also be used as information for identifying the type of base, as in the case of the metal cluster described above.
Labeled oligomers for labeling nucleotide A _{L A:} phosphor particles labeled nucleotide labeled oligomers for G _{L G} of the light-emitting central wavelength 400 nm (purple): phosphor particles labeled nucleotide labeled oligomers for T L of the emission center wavelength of 500 nm (green) _T : phosphor particle with emission center wavelength of 600 nm (orange) Labeled oligomer L _C for label base _C : phosphor particle with emission center wavelength of 700 nm (red)

  The procedure for binding metal clusters or inorganic phosphor particles to the labeled oligomer L is first to use the metal clusters or inorganic phosphor particles as amino compounds having primary or secondary amino groups (for example, monoamino undecagold). Then, the base in the labeled oligomer L is liberated by the action of an appropriate base releasing means to form a hemiacetal-type ribose residue, and then the base is subjected to a reductive amination reaction with an amino compound with respect to the hydroxyl group at the base releasing site. An amino compound is introduced at the position where is bound (see Patent Document 1). After the introduction of the amino compound, the labeled oligomer L is preferably purified to 99.9% or more by various high purity separation and purification techniques.

2. Addition of Adapter Sequence and Immobilization An adapter sequence is added to the 5 ′ end of the target ssDNA and immobilized on the surface of the solid phase (see FIG. 3). Here, an example will be described in which the base sequence of the target ssDNA is determined from the 3 ′ end, but when it is desired to determine from the 5 ′ end, the adapter sequence is added to the 3 ′ end of the target ssDNA.

  As the target ssDNA, one prepared from genomic DNA or artificial double-stranded DNA can be used, and cDNA can also be used. Genomic DNA or artificial double-stranded DNA is fragmented (divided) by DNase treatment, ultrasonic treatment, stirring treatment, etc., and then heated to about 95 ° C. to be denatured into single strands.

  In the base-associated labeling method and the base sequence information acquisition method according to the present invention, the target single-stranded nucleic acid includes not only DNA but also mRNA, tRNA, rRNA, siRNA, miRNA, and shRNA. .

  For immobilization of the target ssDNA on the solid phase surface, for example, a method of biotinylating the target ssDNA and binding it to the slide glass surface using an avidin-biotin bond can be employed. The solid phase may be microbeads, membranes or filters.

3. Labeling of nucleic acid in target ssDNA by CDC method [hybridization]
The solid-phased target ssDNA is brought into contact with the double-stranded library for conversion to perform a hybridization reaction (see FIG. 4). The reaction is carried out under conditions that allow the target ssDNA and the conversion duplex to bind to form a duplex. In the figure, the base sequence shown as 3′-x ₁ -x ₂ -x ₃ -x ₄ -x ₅ -x ₆ -5 'is a base to be identified in the target ssDNA, and the base x is determined according to the procedure described below. ₁ is labeled first, and the bases x ₂ , x ₃ , x ₄ ... X _k (k is an arbitrary integer) are sequentially labeled by repeating it.

[Circularization]
A complex of the target ssDNA formed by the hybridization reaction and the double strand for conversion is treated with DNA ligase to circulate the complex (see FIG. 5).

[Restriction enzyme treatment]
A complex of the circularized target ssDNA and the double strand for conversion is treated with a type II restriction enzyme (see FIG. 6). The restriction enzyme binds to the recognition site (base sequence R) in the double strand for conversion, and the nucleotide sequence site (x ₁ -x ₂ -x ₃ -x ₄ -x _{5 in} FIG. 6) to be identified in the target ssDNA. An enzyme having a cleavage site at a position where at least one nucleotide can be excised from the 3 ′ end of −x ₆ ) is used. In the figure, an example is shown in which only the _first labeled base x1 present at the 3 ′ end of the target ssDNA is cut out.

[dissociation]
After the restriction enzyme treatment, the double-stranded forming part of the cut double strand for conversion is dissociated, and the strand of the double strand for conversion that is not linked to the target ssDNA is removed by washing (see FIG. 7). As a result, “converted ssDNA” is obtained in which the _first labeled base x1 present at the 3 ′ end of the target ssDNA is translocated to the 5 ′ end side through the labeled oligomer. This conversion ssDNA, base x ₁ is turned distinguishable labeled indirectly by insertion addition of labeled oligomers L of specific labeling was made in accordance with the type of base x _1.

By repeating the above-described series of steps of hybridization, circularization, restriction enzyme treatment and dissociation, the bases x ₂ , x ₃ , x ₄ ... X _k (k is an arbitrary integer) are sequentially labeled. Insert and add oligomers. As a result, converted ssDNA having a repetitive sequence having a labeled oligomer and a base x labeled with the labeled oligomer as one repeating unit is generated at the 5 ′ end.

FIG. 8 schematically shows an example of the converted ssDNA generated. In the figure, L _A , L _G , L _T , and L _C indicate labeled oligomers in which specific labels for each base of A, G, T, and C are formed by colloidal gold. The figure "labeled oligomer _{L A} and base A"3'-5'direction,"labeled oligomer _{L G} and base G", "labeled oligomer _{L T} and bases T", "labeled oligomer _{L C} and base C" The repeating unit is shown. The sequence order of each base of A, G, T, and C in the repetitive sequence is the same as that of the target ssDNA, and the converted ssDNA maintains the base sequence information of the target ssDNA.

  The reading accuracy of the base sequence of the target ssDNA depends on the labeling rate of the base in the target ssDNA, and the labeling rate of the base in the target ssDNA is equal to the labeling rate of the labeled oligomer L. For this reason, it is preferable to use the labeled oligomer L having a labeling rate of 99.9% or more using a high-purity separation / purification technique after labeling the inorganic phosphor particles and / or inorganic phosphor particles.

4). Acquisition of base sequence information As described above, the converted ssDNA is obtained by transferring the base information of each base to the labeled oligomer L in a base-specific and high-precision manner while maintaining the base sequence information of the target ssDNA. Accordingly, by identifying the label of the labeled oligomer in each repeating unit in the converted ssDNA, the type of base in the same unit can be determined, and the base sequence of the target ssDNA can be determined.

  The base sequence is determined by the particle size of the colloidal gold labeled with the labeled oligomer L or the particle size of the inorganic phosphor particles labeled with the labeled oligomer L and / or the high resolution and high resolution of the electron microscope. This can be done by directly reading the emission wavelength.

  As described above, according to the base-corresponding labeling method of the present invention, a labeled oligomer L specifically labeled according to the type of base can be inserted and added to each nucleotide of the target ssDNA, and the label is labeled under an electron microscope. By identifying, it is possible to obtain base sequence information of the target ssDNA.

  In the base-corresponding labeling method according to the present invention, the base constituting the nucleotide can be indirectly labeled by reliably inserting the labeled oligomer L at the 3 ′ end or 5 ′ end of each nucleotide of the target ssDNA. In addition, since the labeled oligomer L can be highly purified in advance, the base with a higher labeling rate compared to the conventional method (Patent Document 1) in which the labeling rate is limited directly by chemically labeling the base in the target ssDNA. Can be labeled. Specifically, in the conventional direct method, the labeling rate cannot exceed 95% in terms of chemical reaction yield. However, in the indirect method according to the present invention, the labeled oligomer L can be separated and purified in advance, so that the labeling rate is 99. It can be 9% or more.

  Further, in the conventional method of directly labeling the base in the target ssDNA with metal clusters or inorganic phosphor particles, due to the steric hindrance due to the size of the metal raster and the inorganic phosphor particles with respect to the distance between the bases of the target ssDNA, In some cases, the labeling rate decreased. On the other hand, in the base-corresponding labeling method according to the present invention, in order to reliably insert the labeled oligomer L previously labeled with a metal cluster or inorganic phosphor particles between the nucleotides of the target ssDNA, Steric hindrance does not occur.

  Furthermore, in the base-corresponding labeling method according to the present invention, the label oligomer L itself inserted at the 3 ′ end side or the 5 ′ end side of each nucleotide of the target ssDNA is directly labeled for base identification. Compared with the conventional method (Patent Document 4) in which a beacon labeled with a fluorescent reagent for base discrimination is further hybridized to the oligonucleotide thus prepared, there is no reaction error associated with hybridization, so that the base can be detected at a higher labeling rate. Labeling can be performed. Furthermore, in the conventional method, a variation sequence for specifically hybridizing the oligonucleotide and one of four types (A, G, T, C) beacons is provided in the base sequence of the oligonucleotide. I needed it. In contrast, in the base-corresponding labeling method according to the present invention, since hybridization is unnecessary, the base sequence of the double-stranded part of the labeled oligomer L can be limited to one type.

  According to the base-corresponding labeling method of the present invention, the bases in the nucleic acid can be indirectly labeled at a high labeling rate while maintaining the base sequence information of the nucleic acid. Therefore, the present invention can be used in a sequencing technique for identifying a base through a label under an electron microscope and acquiring nucleic acid base sequence information, and can contribute to simplification and speed-up of the technique.

Claims (10)

  A method for labeling a base in a single-stranded nucleic acid while maintaining the base sequence information of the nucleic acid, depending on the type of base constituting the nucleotide on the 3 ′ end side or 5 ′ end side of the nucleotide. A method for labeling corresponding to a base, comprising a step of inserting an oligonucleotide having a specific label.   The base-corresponding labeling method according to claim 1, wherein the oligonucleotide is inserted by a circular DNA conversion method.   The base-compatible label according to claim 1 or 2, wherein the oligonucleotide labeled with a metal cluster having an average particle diameter of 1-1000 nanometers and / or inorganic phosphor particles having an average particle diameter of 0.5-1000 nanometers is used. Method.   4. The base-corresponding labeling method according to claim 3, wherein the four types of metal clusters having different average particle diameters are used.   The base-corresponding labeling method according to claim 4, wherein the metal cluster is a gold colloid.   The base-corresponding labeling method according to claim 3, wherein four types of the inorganic phosphor particles having different average particle diameters and / or emission wavelengths are used.   The base corresponding | compatible labeling method as described in any one of Claims 1-6 using the oligonucleotide isolate | separated and purified after the said inorganic fluorescent substance particle | grains and / or the said inorganic fluorescent substance particle | grains as said oligonucleotide.   The base sequence information is obtained by identifying the label of the oligonucleotide under an electron microscope for the single-stranded nucleic acid labeled by the base correspondence labeling method according to any one of claims 1 to 7. Method.   A single-stranded nucleic acid in which an oligonucleotide having a specific label according to the type of base constituting the nucleotide is inserted at the 3 ′ end or 5 ′ end of the nucleotide.   The single-stranded nucleic acid according to claim 9, wherein the label is a metal cluster having an average particle diameter of 1 to 1000 nanometers and / or an inorganic phosphor particle having an average particle diameter of 0.5 to 1000 nanometers.

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