JP2003116581A - Labeled nucleic acid and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To use a β-diketone europium complex for detecting a nucleic acid in a region at a low concentration which cannot be detected with a conventional fluorescent dye in a DNA chip or a DNA microarray and to provide a method for assaying an immobilized DNA immobilized on a stationary phase such as a glass or a membrane. SOLUTION: A labeling substance represented by the general formula (I) is bound through a specific connecting group to the nucleic to provide a labeled nucleic acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to labeled nucleic acids effective for highly sensitive and quantitative detection of nucleic acids, and detection of nucleic acids using them.

[0002]

2. Description of the Related Art Radioactive substances have been widely used to detect various biological substances such as nucleic acids. However, although labeling with radioactive isotopes is highly sensitive, it has the drawback of being dangerous during storage, use and processing. On the other hand, fluorescent substances are widely used in the fields of medicine, basic research, etc. as labeling substances because of their ease of handling. For labeling nucleic acids, compounds such as fluorescein, rhodamine, dansyl and pyrene are generally used. In addition, fluorescent substances that intercalate between the two strands of nucleic acid are also used for fluorescent staining. However, the fluorescent substance has a low quantum yield, is affected by other endogenous fluorescent substances, has a small wavelength difference between the excitation light and the fluorescent wavelength, and
A compound having properties such as a poor / N ratio is included.

On the other hand, at present, one of the gene analysis techniques using a fluorescent substance is a DNA chip or a DNA microarray. This method involves immobilizing thousands of genes on a stationary phase such as glass and deriving from mRNA derived from biological samples.
Hybridize the cDNA. At this time, since the cDNA is labeled with a fluorescent substance, the expressed gene pattern on the stationary phase can be specified. Cy3 and Cy5 are generally used as the fluorescent substance of the DNA label. However, these fluorescent substances have a short wavelength difference between excitation light and fluorescence, and cannot repeat measurement because of self-quenching.
It also has a problem that it does not have sensitivity enough to detect a gene having a low expression level. Therefore, if it is possible to use a fluorescent substance that is more sensitive and stable, it is possible to analyze genes with low expression levels and obtain more detailed gene expression pattern data.

Further, in the DNA chip and the DNA microarray, the gene group immobilized on the stationary phase has not been finally quantified, and it is difficult to accurately estimate the difference in the expression level between the genes. . Therefore, if Cy3 and Cy5, which are currently used for DNA chip analysis, and a highly sensitive fluorescent substance having different excitation light and fluorescence wavelength are present, it becomes possible to quantify the gene on the stationary phase.

At present, among more sensitive fluorescent labeling methods, labeling with a complex containing an inorganic ion, particularly labeling with a rare earth ion complex is known. The rare earth ion complex has a long fluorescence lifetime (compared with the fluorescence lifetime of ordinary fluorescent substances of several nanoseconds, the rare earth fluorescent complex has a fluorescence lifetime of tens or hundreds of microseconds or more), and a large excitation / fluorescence wavelength difference. It has a fluorescent characteristic that it has a sharp fluorescent peak. Taking advantage of this characteristic, a time-resolved fluorescence measurement method using a rare earth fluorescent complex as a label has been developed. Due to such characteristics, the time-resolved fluorescence measurement can be used to remove the interference caused by the excitation light and the short-lived background fluorescence derived from the biological sample, thereby enabling highly sensitive measurement.

Up to now, β-diketone complexes and aromatic amine complexes have been known as fluorescent labeling agents using rare earth ion complexes. One of the rare earth fluorescent labeling agents already commercialized is "DELFIA" (Dissociation-Enhanced Lanthanide Flu) developed by Wallac berthold (Finland).
oroimmunoassay) system. In this DELFIA system, isothiocyanato-phenyl-EDTA or isothiocya
natophenyl-DTTA (DTTA = diethylenetriaminetetracetic
acid) and a rare earth complex as a labeling agent to label proteins and nucleic acids, and before measuring fluorescence, β-diketone-trioctylphosphine oxide (TOPO) -Triton X
Fluorescence measurement by adding a so-called fluorescence-enhancing solution containing -100 to liberate the rare earth metal ion from the non-fluorescent complex and generate a micelle solution of β-diketone-rare earth ion-TOPO ternary complex Is (E. Soin
i, T. Lovgren, CRC Crit. Rev. Anal. Chem., 18, 105
-154 (1987)). However, in this DELFIA system, since excess β-diketone and TOPO are present in the measurement solution, they may react with rare earth metal ions from the environment and emit strong fluorescence, so they are extremely susceptible to contamination with rare earth metal ions. It has the big drawback of being easy. Furthermore, the DELFIA system requires the addition of a fluorescence enhancing solution, which has the drawback of having many reaction steps and the drawback of not being able to perform solid phase measurement.

Another commercially available rare earth fluorescent labeling agent is a labeling agent used in the FIAGEN system developed by Diamandis et al. (Canada) (EP Diamandis, i).
n Biochem., 21, 139-150 (1988); EFG Dickson,
A. Pollak, EP Diamandis, Pharmac. Ther., 66, 207
-235 (1995)). The FIAGEN system is a fluorescent europium complex (4,7-bis (chlorosu
lfophenyl) -1,10-phenanthroline-2,9-dicarboxylic ac
This is a measurement method using id (BCPDA) -Eu ³⁺ ). This system does not have the problem of Europium contamination from the environment and is also capable of solid phase measurements. However, since the fluorescence intensity of the labeling agent in this system is weaker than that of the DELFIA system by two digits or more, it has a drawback that detection sensitivity is low and high-sensitivity analysis is difficult.

Therefore, it has already been found by Matsumoto et al. That the fluorescence intensity of the β-diketone-europium complex is enhanced by placing an electron-withdrawing substituent adjacent to the β-diketone. Based on this finding, Matsumoto et al. Developed a β-diketone type fluorescent complex compound that forms a complex with europium ion and exhibits a long fluorescence lifetime and high fluorescence intensity,
Its usefulness has been shown (JP-A-9-241233 and Analytical Chemistry, 1998, Volume 70, 596-601).
page). Furthermore, Matsui et al. Attempted to detect on a biological sample by introducing the β-diketone-type fluorescent complex into a nucleic acid in order to use it for labeling a nucleic acid (Japanese Patent Laid-Open No. 2000-19171).

However, Japanese Patent Laid-Open No. 2000-19171 has not sufficiently examined the sensitivity and quantitativeness of labeling nucleic acid. Further, it has not been reported that the β-diketone type europium complex was used for a DNA chip or a DNA microarray. Further, JP-A-2000-19171 has low nucleic acid labeling efficiency and is not suitable for a DNA chip and a DNA microarray which actually use an extremely small amount of sample.

[0010]

From the background described above, the object of the present invention is to increase the labeling efficiency of nucleic acids by using a β-diketone type europium complex, and it is not possible to detect with conventional fluorescent dyes in DNA chips or DNA microarrays. Use for the purpose of detecting nucleic acids in the low concentration range that was possible, and DNA immobilized on a stationary phase such as glass or membrane.
It is to provide a method for quantifying.

[0011]

In order to solve the above problems, the inventors of the present invention have conducted extensive studies to find a highly sensitive nucleic acid labeling method using a β-diketone type europium complex, and as a result, have found that a specific linking group It has been found that, when a nucleic acid is labeled via, a highly sensitive label suitable for labeling a small amount of sample used for a DNA chip can be obtained. In addition, we were able to gain new knowledge about the use of labeled nucleic acids. The β-diketone europium complex is highly sensitive and chemically stable, and can be used in combination with the fluorescent dye currently used in the DNA chip. Therefore, the fluorescent complex can be efficiently introduced into nucleic acid. The inventors have completed the present invention by finding a method, a method for labeling a small amount of sample used in a DNA chip, and a method for quantifying a gene group on a DNA chip. That is, the present invention includes the following (1) to
Provide (31).

(1) General formula (I)

[Chemical 25] [In formula, n shows the integer of 1-6. ] The labeled body represented by the following general formula (II)

[Chemical formula 26] [In the formula, a represents an integer of 1 to 10, and X represents a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the base, phosphate, sugar moiety or nucleic acid of the nucleic acid. ] Or general formula (III)

[Chemical 27] [In the formula, b, d, and f are the same or different and represent an integer of 0 to 10, and c and e are the same or different and are 0.
Alternatively, it represents an integer of 1. However, b, d, and f are all 0
Not included if X represents a base of a nucleic acid, a phosphate, a sugar moiety or a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the nucleic acid. ] The labeled nucleic acid couple | bonded with the nucleic acid through the linking group which has the amino group shown by these.

(2) General formula (I)

[Chemical 28] [In formula, n shows the integer of 1-6. ] The labeled compound represented by the following formula (IV)

[Chemical 29] [Wherein R 1, R 2 and R 3 are the same or different and each represents a hydrogen atom, or an aminoalkyl group represented by the above general formula (II) or (III), and Y represents a base of a nucleic acid, a phosphate, or a sugar moiety. Represent ] The labeled nucleic acid couple | bonded with the nucleic acid through the coupling group shown by these.

(3) General formula (I)

[Chemical 30] [In formula, n shows the integer of 1-6. ] The labeled compound represented by the formula [V] is a labeled nucleic acid bound to a nucleic acid via a linking group composed of aminoalkyl.

[Chemical 31] [In the formula, N represents either a 2'-deoxythymidine nucleotide, a 2'-deoxyadenine nucleotide, a 2'-deoxycytosine nucleotide or a 2'-deoxyguanine nucleotide, n is an integer of 1 to 6, and i is 1 The integer of -10, q represents the integer of 1-100. ] The said labeled nucleic acid shown by these.

(4) General formula (I)

[Chemical 32] [In formula, n shows the integer of 1-6. ] The labeled compound represented by the formula [V] is a labeled nucleic acid bound to a nucleic acid via a linking group composed of aminoalkyl.

[Chemical 33] [In the formula, N represents any one of a uridine nucleotide, an adenine nucleotide, a cytosine nucleotide, or a guanine nucleotide, n is an integer of 1 to 6, and i is 1 to 10
, And q represents an integer of 1 to 100. ],
The labeled nucleic acid.

(5) General formula (I)

[Chemical 34] [In formula, n shows the integer of 1-6. ] The label | marker shown by these is couple | bonded with a nucleic acid through a coupling group, (1)-(4)
A method for producing the labeled nucleic acid according to any one of 1.

(6) A method for amplifying a nucleic acid, which uses the labeled nucleic acid according to any one of the above (1) to (4) as a probe. (7) A method for isolating a reaction product by amplifying the nucleic acid using the labeled nucleic acid according to any one of (1) to (4) above as a probe and then removing an excess of the primer from the reaction product. (8) A reverse transcription reaction using the labeled nucleic acid according to any one of (1) to (4) above as a primer. (9) A reaction product obtained by the reverse transcription reaction described in (8) above. (10) After adding a heavy metal ion to the labeled nucleic acid described in (1) to (4) above to form a fluorescent complex, it is reacted with a target substance on a biological sample, and the fluorescence of the fluorescent complex after the reaction is measured. A method for analyzing a nucleic acid, which is characterized. (11) The labeled nucleic acid according to any one of (1) to (4) above is reacted with a target substance on a biological sample, a heavy metal ion is added, and the fluorescence of the resulting fluorescent complex is measured. A method for analyzing a nucleic acid. (12) A fluorescent complex containing the labeled nucleic acid according to any one of (1) to (4) above and a heavy metal ion. (13) The fluorescent complex according to the above (12), wherein the heavy metal ion is a lanthanoid metal ion or a radium ion. (14) The lanthanoid metal ion is europium,
The fluorescent complex according to (13) above, which is an ion of samarium, terbium, or dysprosium or a mixture thereof. (15) The labeled nucleic acid according to any of (1) to (4) above, which is reversibly or irreversibly immobilized on a stationary phase. (16) A method for quantifying a nucleic acid on a stationary phase by adding a heavy metal ion to the labeled nucleic acid according to (15) above and measuring the fluorescence of the resulting fluorescent complex. (17) By adding a heavy metal ion to the labeled nucleic acid according to any one of (1) to (4) above, it is reversibly or irreversibly immobilized on a stationary phase, and the fluorescence of the resulting fluorescent complex is measured. , A method for quantifying nucleic acids on a stationary phase.

(18) General formula (VI)

[Chemical 35] Or general formula (VII)

[Chemical 36] [Wherein R ₁ and R ₂ are each a 2′-deoxythymidine nucleotide, a 2′-deoxyadenine nucleotide, a 2′-deoxycytosine nucleotide, a 2′-deoxyguanine nucleotide, a general formula (VI) or a general formula (VII). Label-bound 2'-deoxyoligoribonucleotide consisting of 2'-deoxyuridine derivative, n is an integer of 1 to 6, and h and m are the same or different and are an integer of 1 to 6. ] The 2'-deoxy polyribonucleotide which has the label | marker coupling | bonding 2'-deoxy uridine derivative shown by these in at least 1 residue chain.

(19) The following general formula (VIII)

[Chemical 37] Or the following general formula (IX)

[Chemical 38] [H and m are the same or different and each represents an integer of 1 to 6. ] A deoxypolyribonucleotide containing at least one compound represented by the formula:

[Chemical Formula 39] [In formula, n shows the integer of 1-6. ] The method for producing a 2'-deoxypolyribonucleotide according to the above (18), which comprises binding the labeled compound shown in

(20) The 2'-deoxypolyribonucleotide described in (18) above is reacted with a target substance on a biological sample, a heavy metal ion is added, and the fluorescence of the resulting fluorescent complex is measured. Method for analysis of target substance. (21) The purpose of measuring the fluorescence of a fluorescent complex produced by adding a heavy metal ion to the 2′-deoxypolyribonucleotide according to (18) above and then reacting the target substance on a biological sample. Analytical method for substances.

(22) General formula (X)

[Chemical 40] [In the formula, h is 1 to 6, e is 1 to 10, and n is an integer of 1 to 6. ] 2′-deoxyuridine-
5'-triphosphoric acid. (23) The above (22) in which e is 5, h is 1, and n is 3.
Labeled body-bound 2'-deoxyuridine-5'-triphosphate as described.

(24) (i) General formula (XI)

[Chemical 41] [In the formula, e represents an integer of 1 to 10], the aminocarboxylic acid derivative represented by the general formula (I)

[Chemical 42] [In formula, n shows the integer of 1-6. ] The labeled compound represented by the formula is reacted to give a compound of formula (XII)

[Chemical 43] [Wherein, e is the same as the above], and a labeled body-bound aminocarboxylic acid body is obtained, and (ii) the labeled body-bound aminocarboxylic acid body is prepared from succinimide and 1- (3-dimethylaminopropyl). -3-ethylcarbodiimide hydrochloride in the presence of to obtain formula (XIII),

[Chemical 44] (Iii) Next, the general formula (VIII)

[Chemical formula 45] [H represents an integer of 1 to 6] ] The method of manufacturing the label | marker coupling | bonding 2'-deoxyuridine-5'-triphosphoric acid of Claim 22 by performing a condensation reaction with the compound shown by these.

(25) General formula (XIV)

[Chemical formula 46] [In the formula, m represents 1 to 6, e represents 1 to 10, and n represents an integer of 1 to 6. ] 2′-deoxyuridine-
5'-triphosphoric acid.

(26) The label-bonded 2'-deoxyuridine-5 'according to the above (25), wherein e is 5, m is 1, and n is 3.
-Triphosphoric acid.

(27) General formula (XIII)

[Chemical 47] [In formula, e represents 1-10, n represents the integer of 1-6. ] The compound shown by the general formula (IX)

[Chemical 48] [M represents an integer of 1 to 6] ] It is made to react with the compound shown by these, The manufacturing method of 2'-deoxyuridine-5'-triphosphoric acid couple | bonded with the label | marker as described in said (25).

(28) A method for labeling a nucleic acid using the label-bound 2'-deoxyuridine-5'-triphosphate according to the above (22) or (25). (29) A labeled nucleic acid obtained by the method according to (28) above. (30) A method for analyzing a target substance, which comprises reacting the labeled nucleic acid according to (29) above with a target substance on a biological sample, adding a heavy metal ion, and measuring the fluorescence of the generated fluorescent complex. (31) A method for analyzing a target substance, which comprises adding a heavy metal ion to the labeled nucleic acid according to (29) above and then reacting the target substance with the target substance on a biological sample, and measuring the fluorescence of the generated fluorescent complex.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention first provides a labeled nucleic acid in which the labeled compound represented by the general formula (I) is bound to a nucleic acid via a specific linking group.

[0028]

[Chemical 49] [In formula, n shows the integer of 1-6. The label represented by the general formula (I) is a known nucleic acid label, for example, Yua
n, J. and Matsumoto, K. Anal. Chem. (1998) 70, 596-
It can be obtained by the method described in 601. Particularly preferred label in the present invention is 4,4′-bis (1 ″, 1 ″, 1 ″, 2 ″, 2 ″, 3 ″, 3 ″ -heptafluoro shown in FIG. -Four'',
6 ''-hexanedione-6 ''-yl) chlorosulfo-o-terphenyl (4,4'-bis (1 '', 1 '', 1``, 2 '', 2``, 3 '' , 3``-he
ptafluoro-4``, 6 ''-hexanedion-6 ''-yl) chlorosulfo-
o-terphenyl) (BHHCT).

The linking group essentially used in the present invention is a linking group represented by the general formula (II) or the general formula (II
A linking group represented by I) and a linking group represented by the general formula (IV).

[0030]

[Chemical 50] [In the formula, a represents an integer of 1 to 10, and X represents a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the base, phosphate, sugar moiety or nucleic acid of the nucleic acid. ]

[Chemical 51] [In the formula, b, d, and f are the same or different and represent an integer of 0 to 10, and c and e are the same or different and are 0.
Alternatively, it represents an integer of 1. However, b, d, and f are all 0
Not included if X represents a base of a nucleic acid, a phosphate, a sugar moiety or a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the nucleic acid. ]

[Chemical 52] [Wherein R 1, R 2 and R 3 are the same or different and each represents a hydrogen atom, or an aminoalkyl group represented by the above general formula (II) or (III), and Y represents a base of a nucleic acid, a phosphate, or a sugar moiety. Represent ]

In all of these, a primary amino group is bonded to a primary hydrocarbon, and the steric bulkiness of the site that binds to a labeled substance such as BHHCT is low, the reactivity is high, and the introduction of a fluorescent dye is easy. is there. Further, it can be easily introduced into a nucleic acid, and a plurality of labels can also be introduced by branching as in the general formula (IV).

In the present invention, "nucleic acid" means nucleoside, mononucleotide, dinucleotide, trinucleotide, 2'-deoxynucleoside, 2'-deoxymononucleotide, 2'-deoxydinucleotide, 2'-deoxytrinucleotide. 2 nucleotides or more
It refers to RNA and DNA consisting of 2'-deoxynucleotides and natural nucleic acid analogs with modified base or sugar moieties.

The labeled nucleic acid can be obtained by binding the labeled body represented by the general formula (I) and the linked body with a sulfonamide bond or an amide bond. The labeled nucleic acid of the present invention can be used, for example, as a primer in a PCR reaction to specifically amplify a nucleic acid, and a reaction product (amplification product) can be detected with high sensitivity,
And it can be detected quantitatively. When used as a primer, the length of the nucleic acid is preferably about 10 to 100 bases.

After the nucleic acid is amplified as described above, it is possible to quantitatively detect the reaction product by removing excess primer from the reaction product and isolating the reaction product prior to the fluorescence measurement. Is preferred. The labeled nucleic acid of the present invention can also be used as a primer for a reverse transcription reaction, and the reaction product of the reverse transcription reaction can be detected with high sensitivity and quantitatively.

In the fluorescence measurement, heavy metal ions are added to the labeled nucleic acid of the present invention to form a fluorescent complex, which is then reacted with a target substance on a biological sample and the fluorescence of the fluorescent complex after the reaction is measured, or The labeled nucleic acid of the present invention may be reacted with a target substance on a biological sample, and then a heavy metal ion may be added to measure the fluorescence of the resulting fluorescent complex, but it is not particularly limited.

In the present invention, the "biological sample" means
A virus, a microorganism, a bacterium, a plant, an animal, or a tissue or a part of a tissue thereof, or a cell thereof, which is a DNA chip or D depending on the mode of use of the labeled nucleic acid of the present invention.
It may be a nucleic acid immobilized on the NA array. In the present invention, the “target substance” means a nucleic acid to be detected using the labeled nucleic acid of the present invention, and a nucleic acid that can hybridize with the labeled nucleic acid of the present invention. The target substance is, for example, a nucleic acid serving as a template in a PCR reaction or a reverse transcription reaction, a nucleic acid on a DNA chip or a DNA array, or a biological sample reacted with the DNA chip or the DNA array, depending on the mode of use of the labeled nucleic acid of the present invention. Nucleic acid contained therein.

The heavy metal ions preferably used in the present invention are lanthanoid metal ions such as europium, samarium, terbium and dysprosium, or radium ions, or a mixture thereof. The labeled nucleic acid of the present invention can be used after being immobilized reversibly or irreversibly on a stationary phase such as glass, membrane and beads. The immobilization of the nucleic acid on the stationary phase can be performed by a means commonly used in the art. On the other hand, the labeled nucleic acid of the present invention can be used for detection of the target substance by hybridizing with the nucleic acid which is the target substance immobilized on the stationary phase.

The nucleic acid on the stationary phase is detected by reversibly or irreversibly immobilizing the labeled nucleic acid of the present invention on the stationary phase, adding the above heavy metal ions, and measuring the fluorescence of the resulting fluorescent complex. , Can be done quantitatively. Alternatively, it can be quantitatively detected by adding a heavy metal ion to the labeled nucleic acid of the present invention, then reversibly or irreversibly immobilizing it on a stationary phase, and measuring the fluorescence of the resulting fluorescent complex. . The present invention also provides a 2'-deoxypolyribonucleotide having a label-bound 2'-deoxyuridine derivative represented by the general formula (VI) or the general formula (VII) in at least one residue chain.

[0039]

[Chemical 53]

[Chemical 54] [Wherein R ₁ and R ₂ are each a 2′-deoxythymidine nucleotide, a 2′-deoxyadenine nucleotide, a 2′-deoxycytosine nucleotide, a 2′-deoxyguanine nucleotide, a general formula (VI) or a general formula (VII). Label-bound 2'-deoxyoligoribonucleotide consisting of 2'-deoxyuridine derivative, n is an integer of 1 to 6, and h and m are the same or different and are an integer of 1 to 6. ]

The above 2'-deoxypolyribonucleotide is prepared by converting the labeled form represented by the general formula (I) into the general formula (VII
It can be obtained by binding I) or a compound represented by the general formula (IX) (modified base) to a deoxypolynucleotide containing at least one.

[0041]

[Chemical 55]

[Chemical 56] [H and m are the same or different and each represents an integer of 1 to 6. ]

The above 2'-deoxypolyribonucleotide is reacted with a target substance on a biological sample, and then a heavy metal ion is added to measure the fluorescence of the resulting fluorescent complex. The target substance can be analyzed by reacting it with the target substance on the sample and measuring the fluorescence of the resulting fluorescent complex.

The present invention further includes a label-bonded 2'-deoxyuridine-represented by the general formula (X) or the general formula (XIV).
Provides 5'-triphosphoric acid. The label-bound 2'-deoxyuridine-5'-triphosphate can be produced specifically as described in the following Examples and can be used for labeling various nucleic acids.

[0044]

[Chemical 57] [In the formula, h is 1 to 6, e is 1 to 10, and n is an integer of 1 to 6. ]

[Chemical 58] [In the formula, m represents 1 to 6, e represents 1 to 10, and n represents an integer of 1 to 6. ]

The nucleic acid labeled with the label-bound 2′-deoxyuridine-5′-triphosphate is reacted with a target substance on a biological sample, a heavy metal ion is added, and the fluorescence of the resulting fluorescent complex is measured. Alternatively, the target substance can be analyzed by adding a heavy metal ion to the labeled nucleic acid, reacting the target substance with the target substance on the biological sample, and measuring the fluorescence of the generated fluorescent complex.

In the DNA chip currently used, P
A CR-amplified cDNA library or synthetic oligonucleotide is immobilized on a substrate (glass). However, since several washing treatments are carried out after the immobilization of the DNA, it has not been possible to measure the amount of the gene that the DNA is missing from the substrate and is finally immobilized on the substrate. In the current assay of prepared DNA chips, fluorescence-labeled cDNA is hybridized with DNA on a substrate and assayed by a fluorescence intensity pattern. However, since the amount of the fluorescently labeled cDNA is not constant among the genes, accurate quantification has not been performed. Therefore, by directly and directly quantifying the DNA immobilized on the substrate, a more accurate product assay of the DNA chip becomes possible, and further, a high-quality DNA chip capable of comparing the relative expression levels between genes. Can be supplied.

DNA labeled with a highly sensitive fluorescent dye
After immobilizing the
Since it was possible to quantify A, DNA was labeled with a highly sensitive fluorescent dye. First, an oligonucleotide labeled with a highly sensitive fluorescent dye was synthesized. Then, PCR was performed using the fluorescently labeled synthetic oligonucleotide as a primer. Β as a highly sensitive fluorescent compound
-It was decided to use a diketone type fluorescent complex. Since the β-diketone type fluorescent complex emits fluorescence having a long life in the presence of rare earth metal ions, highly sensitive fluorescence measurement can be performed by performing time-resolved fluorescence measurement.

By using the highly sensitive fluorescent compound-labeled oligonucleotide as a primer and measuring the fluorescence, it becomes possible to quantify the DNA immobilized on the substrate,
A more rigorous DNA chip assay can be performed. The assayed DNA chip is hybridized with a fluorescent (often Cy3 and Cy5) labeled biological sample, and can be used for studying the actual gene expression level. The excitation light of the β-diketone-type fluorescent complex used this time is the above-mentioned fluorescent compound (Cy3) used for labeling the biological sample.
Since it does not overlap with Cy5), it does not affect the measurement of the biological sample. Further, by washing the glass after the end of the assay with another complex compound such as EDTA, the rare earth metal ion is released from the β-diketone type fluorescent complex and β
-It is also possible not to emit fluorescence derived from a diketone type fluorescent complex. Figure 9 shows the immobilized DN on the DNA chip.
A scheme of quantifying A and analyzing a biological sample using a DNA chip after the assay is shown. The result of PCR fluorescence measurement is
As shown in FIG.

Immobilization of synthetic oligonucleotides on a substrate is also necessary for producing a good quality DNA chip. PCR at the end of synthetic oligonucleotide
It is possible to quantify the immobilized synthetic oligonucleotide by measuring the fluorescence after the β-diketone type fluorescent complex is bound and immobilized in the same manner as the primer.

[0050]

[Examples] Thin layer chromatography was performed on Kieselgel 60F254 plate (Merck) using chloroform-methanol as a developing solvent. Wakogel C-200 or C-300 (Wako Pure Chemical Industries) was used for column chromatography. UV-visible spectrum is Shimadzu UV-2500PC
It measured using the spectrophotometer.

¹ H-NMR was measured using JEOL JNM-EX270 with tetramethylsilane as an internal standard. ³¹ P-
NMR was measured using JEOL JNM-EX270 with inorganic phosphoric acid as an internal standard. Mass spectrometry (FABHRMS) is JEOLJ
It was measured using MS-HX110 or JMS-700TZ. Oligonucleotide synthesis by Applied Biosystems 3
It was performed on a 94 type DNA / RNA synthesizer.

Gilson's apparatus was used for HPLC, and the analysis was
This was done using a Waters 996 photodiode array detector. Waters μ Bondasphere as a column for reversed-phase analysis
C18, 300Å (inner diameter 3.9 mm x length 150 mm) or μBondasphere C8, 300Å (inner diameter 3.9 mm x length 1)
50 mm), GL Science Inertsi as a column for reverse phase separation
l ODS-3 C18 (inner diameter 8.0 mm x length 300 mm), Tosoh TSK-GEL DEAE-for anion exchange analysis
2SW (inner diameter 4.6 mm × length 250 mm) was used. The mobile phase used was a gradient of acetonitrile in 0.1 M triethylammonium acetate buffer (TEAA, pH 7.0) for the reverse phase and 20% ammonium formate in acetonitrile in the case of anion exchange.

For fluorescence measurement, Wallac 1420 A from PERKIN ELMER
RVOsx was used. Applied Biosystems Gene A for PCR
It was performed using the mp PCR system 9700.

Example 1 D used in PCR and reverse transcription reaction
Synthesis of NA Primer Oligodeoxyribonucleotides (1) to which BHHCT shown in FIG. 1 was bound, oligodeoxyribonucleotides bound to fluorescein (2) used as a comparative experiment, and oligodeoxyribonucleotides (3) having no amino group and fluorescein were synthesized. In addition, as a primer used for PCR, oligodeoxyribonucleotide (4)
Was also synthesized. Oligodeoxyribonucleotide (1): 5'-N-CTAGCCATATGTATATCT-3 '(NRT18; N represents 6-amino-1-hexanol phosphate), oligodeoxyribonucleotide (2): 5'-FCTAGCCATATGTATATCT-
3 '(FRT18; F represents fluorescein), oligodeoxyribonucleotide (3): 5'-CTAGCCATATGTATATCT-
3 '(RT18) has the same base sequence (SEQ ID NO: 1). The base sequence of oligodeoxyribonucleotide (4) is 5'-CAAGGAGATGGCGCCCAACAGTCCCCC-3 '(SEQ ID NO: 2). Oligodeoxyribonucleotide (1),
The structure of (2) is shown in FIG.

Each of the above oligodeoxyribonucleotides was prepared by using a deoxynucleoside 3'-phosphoramidite (purchased from Perkin Elmer Japan Applied Biosystems Division) as a raw material and an automatic DNA synthesizer (Model 394A; Perkin Elmer Japan Applied Biosystems Division) 1 μm
It was synthesized on the ol scale. As M (Glen Research) and F, N-monomethoxytrityl-6-aminohexyl phosphoramidite and fluorescein phosphoramidite (Glen Research) were used.

After completion of the synthesis, oligodeoxyribonucleotide (1) and oligodeoxyribonucleotide (3) were treated and purified as follows. The oligodeoxyribonucleotide was cut out from CPG (Controlled Pore Glass) with concentrated ammonia water and heated at 40 ° C. for 40 hours. After distilling off the solvent and dissolving in deionized water, C18
Open column chromatography (manufactured by Waters) was performed (column size 0.8 × 18 cm: 5-40).
% Acetonitrile, 0.1M Triethylammonium acetate; hereinafter referred to as "TEA
A)) elution with a linear concentration gradient using an aqueous solvent). Collect the fractions eluted with about 30% concentration of acetonitrile, add 2 ml of 80% acetic acid aqueous solution,
Stir for 20 minutes. Acetic acid was distilled off under reduced pressure, and the aqueous layer was washed with ethyl acetate. After distilling off the solvent, it was dissolved in 1 ml of sterilized water.

After the synthesis of oligodeoxyribonucleotide (2) was completed, CPG (Controlled Por
The oligodeoxyribonucleotide was cut out from e Glass) and heated at 40 ° C. for 40 hours. The solvent was distilled off and the residue was dissolved in deionized water. Oligodeoxyribonucleotide (2) and oligodeoxyribonucleotide (3)
Was collected by reverse phase HPLC and then ion exchanged HPLC
Was collected and purified. The oligodeoxyribonucleotide (4) was separated and purified only by reverse phase HPLC. Reversed phase HPL
The conditions of C were as follows: Column: μ-Bondasphere (C-18) column Φ3.9 × 150 mm (manufactured by Waters) Solvent: A solution 5% acetonitrile / 0.1M TEAA
(PH 7.0); B solution 25% acetonitrile / 0.1
M TEAA (pH 7.0).

[0058]

[Table 1]

The conditions of the ion exchange HPLC were as follows: Column: TSK-gel DEAE-2SW column Φ 4.6 × 2
50 mm (manufactured by Waters) Solvent: A solution 20% acetonitrile; B solution 20%
Acetonitrile / 2M ammonium formate.

Example 2 Binding reaction of oligodeoxyribonucleotide (1) with BHHCT (Fig. 2) The amount of oligodeoxyribonucleotide (1) (2.87 nmol) was adjusted to 0.
Dissolve in 20 μl of 5 M sodium carbonate buffer (pH 9.5) and add BH
A solution of HCT in acetonitrile (2.86 nmol / μl:
20 μl) was added and the mixture was heated at 50 ° C. After 1 hour, 50 μl of the reaction solution was desalted using a microbio spin column (BIO-RAD) and the reaction solution was evaporated under reduced pressure. The residue was dissolved in 200 μl of sterilized water, and then BHHCT-conjugated oligonucleotide (1) was purified by reverse phase HPLC. Reversed-phase HPLC conditions were as follows: Reversed-phase HPLC column: μ-Bondasphere (C-18) column, Φ.
3.9 × 150 mm (manufactured by Waters) Solvent: A solution 5% Acetonitrile / 0.1M TEAA
(PH 7.0); B solution 50% acetonitrile / 0.1M TEAA (p
H7.0).

BHHCT-binding oligoribonucleotide (1)
Table 2 shows the conditions and retention time of the linear concentration gradient in the reverse phase HPLC of.

[0062]

[Table 2]

After the obtained BHHCT-conjugated oligoribonucleotide (1) was purified, its molecular weight was analyzed by MALDI-TOF / MS. Mass spectrum result: MALDI-TOF / MS theoretical value C ₂₁₂ H ₂₅₁
F ₁₄ N ₆₂ O ₁₁₇ P ₁₈ S (M-H), 6395.3, found 6395.

Example 3 PCR reaction using fluorescein-bound primer and non-dye-bound primer Oligodeoxyribonucleotide (2) (FRT18) or oligodeoxyriboribonucleotide (3) (RT18)
And pQBI using oligodeoxyribonucleotide (4)
PCR was performed using 63 (Takara Shuzo) as a template. pQBI63 (5
ng / μl: 2 μl), oligodeoxyribonucleotide (4) (5 μM; 4 μl), RT18 or FRT18 (5 μM;
4 μl), PCR buffer (x10, 20 mM Mg ²⁺ , attached; 5 μl), dNTP mixture (2.5 mM; 4 μl), sterilized water (30.75 μl), Ex Taq (5 units / μl; 0.25 μl; Takara Shuzo) Mix and bring the total volume to 50 μl, heat at 94 ° C for 5 minutes, then 94 ° C for 60 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds for 2 seconds.
PCR was performed by repeating 5 cycles and heating at 72 degrees for 5 minutes. The product after PCR was a 2% NuSieve agarose gel (NuS
ieve, Takara Shuzo: agarose mixed 2: 1).

Example 4 PCR using BHHCT-conjugated oligodeoxyribonucleotide (1) Example 3 was performed using BHHCT-conjugated oligodeoxyribonucleotide (1) and oligodeoxyribonucleotide (4).
PCR was performed using pQBI63 as a template in the same manner as in. pQBI6
3 (5 ng / μl: 2 μl), oligodeoxyribonucleotide (4) (5 μM; 4 μl), BHHCT-conjugated oligodeoxyribonucleotide (1) (5 μM; 4 μl), PCR buffer (x10, 20 mM Mg ²⁺ , attached) 5 μl), dNTP mixture (2.5 mM; 4 μl), Tween20 (2 μl), sterile water (28.75)
μl) and Ex Taq (5 units / μl; 0.25 μl; Takara Shuzo) were mixed to make the total volume 50 μl, and after heating at 94 ° C. for 5 minutes, 94
25 cycles of 60 seconds at 60 degrees, 30 seconds at 50 degrees, and 30 seconds at 72 degrees were repeated, and PCR was performed by heating at 72 degrees for 5 minutes. PC
The R product was analyzed on a 2% NuSieve agarose gel (NuSieve, Takara Shuzo: agarose mixed 2: 1).

Example 5 Removal of Primer from PCR Product, Immobilization on Immunoplate and Fluorescence Measurement It was carried out using the BHHCT-conjugated oligodeoxyribonucleotide (1) or oligodeoxyribonucleotide (2) obtained in Example 4. 5 from PCR reaction solution (50 μl)
Take μl, add EXO SAP I (2 μl: Pharmacia) and digest at 37 ° C for 15 minutes to decompose excess primer,
The reaction solution was heated at 80 ° C. for 15 minutes to inactivate the enzyme. Total amount of reaction solution (7 μl) or part of it (1 μl, 2 μl,
4 μl) and take PBS (x10; 5 μl), 2% BSA
(2.5 μl) and sterilized water were added to make the total volume 50 μl. The mixed solution was placed on a 96-well immunoplate previously coated with protamine and heated at 37 ° C. 1
After 6 hours, remove the supernatant and use PBST (200 μl) 3 times.
Hybridization solution (50 mM Tris-HCl (pH
7.5), 50 mM sodium chloride; 200 μl), washed twice, and added a buffer solution for time-resolved fluorescence measurement (50 mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 2 μM europium chloride; 50 μl), and the time for fluorescein or europium Degraded fluorescence was measured (Fig. 3).

1.4 or 10% of the PCR product was immobilized on an immunoplate and the fluorescence was measured in the presence of europium ions. BH indicates time-resolved fluorescence derived from BHHCT, F indicates fluorescence derived from fluorescein, and the vertical axis of each graph shows the value divided by the background value. PCR for Primer / ExoSAP I
Only 10% of the BHHCT-conjugated oligodeoxyribonucleotide (1) (50 pmol) used in Example 1 was treated with ExoSAP I, indicating that the fluorescence of the PCR product is not derived from the BHHCT-conjugated oligonucleotide (1). BG is the background value (1.0). The conditions for the fluorescence measurement of fluorescein and europium are as follows.

Fluorescein fluorescence measurement Excitation light wavelength 485 nm Fluorescence wavelength 535 nm counting time 1 second Lamp energy 9728

Eupium fluorescence measurement (time-resolved measurement) Excitation light 355 nm Fluorescence wavelength 615 nm flash energy level 187 counting delay 400 μsec counting window 400 μsec counting cycle 1000 μsec

As a result of the fluorescence measurement, the PCR product using the oligodeoxyribonucleotide (2) showed almost the same intensity as the fluorescence of the measurement buffer solution only (control), but BHHCT-conjugated oligodeoxyribonucleotide (1)
The PCR product using was able to be detected depending on the amount. Therefore, it became clear that the labeled nucleic acid of the present invention can detect PCR products with high sensitivity and quantitatively.

Reference Example 1 Preparation of Polyribonucleotide (5) by In Vitro Transcription Reaction pQBI63 was treated with NheI to form a linear DNA double strand (0.5 μm).
g, 5 μl) in reaction buffer (× 10, (pH 7.5); AmpliS
cribe ^TM T7 transfer kit included; Air Brown) 2μ
l, 25 mM NTP mixed solution (ATP, GTP, CTP,
UTP) 6 μl, 100 mM dithiothreitol 2 μ
1, T7 RNA polymerase solution (AmpliScribe ^™ T7 transcription kit attached; Air Brown Co., Ltd.) 2 μl, sterilized water 3 μl, and the reaction was started at 37 ° C. 2 hours later DNase
1 μl of I (AmpliScribe ^™ T7 transcription kit attached; Air Brown) was added and heated at 37 ° C. for 15 minutes to cleave the template DNA. After desalting with NAP-5, the solvent was distilled off under reduced pressure, followed by extraction with phenol / chloroform and chloroform, followed by ethanol precipitation to obtain polyribonucleotide (5) (SEQ ID NO: 3), which was used in the following reverse transcription reaction. I served.

Example 6 Reverse Transcription Reaction Using Fluorescence-Labeled Oriribonucleotides BHHCT-conjugated oligodeoxyribonucleotides (1) or oligodeoxyribonucleotides (2) were used as primers to prepare polyribonucleotides (5) obtained in Reference Example 1. A reverse transcription reaction was performed (Fig. 4a).

Polyribonucleotide (5) (181 μ
M, 0.55 μl) and primer for reverse transcription BHHCT-conjugated oligodeoxyribonucleotide (1) or oligodeoxyribonucleotide (2) (50 μM, 2.0 μl)
Buffer for reverse transcription (x5, 250 mM Tris-HCl p
H8.3, 375 mM KCl, 15 mM MgCl ₂ , 50 mM DTT: 2.0
μl), heated at 65 ° C. for 2 minutes and then left at room temperature. Then dimethyl sulfoxide (hereinafter DMSO: 1.0
μl), 10 mM dATP (0.5 μl), 10 mM dG
TP (0.5 μl), 10 mM dTTP (0.5 μl), 1
0 mM biotin-conjugated dCTP (biotin-14-dCTP: GIBCO BRL
(0.5 μl), reverse transcriptase M-MLV (200 units / μl,
2.0 μl: Promega) was added and the reaction was carried out at 37 ° C.
As a comparative experiment, BHHCT-conjugated oligodeoxyribonucleotide (1) or oligodeoxyribonucleotide (2) was used as a primer, and 10 m in the above composition was used.
Without M biotin-bound dCTP, 10 mM dCTP (0.
A reaction solution with 5 μl) added instead was also prepared.

One hour later, M-MLV (Moloney Murine Leukem
ia Virus Reverse transcriptase) (200units / μl)
(0.5 μl) was added, and the mixture was further heated for 1 hour. After the reaction, 31 μl of sterilized water, 200 mM EDTA-Na ₂ (12.5 μl),
Add 5N aqueous sodium hydroxide solution (2.0 μl) and add 6
Heated at 5 degrees. After 30 minutes, 1N aqueous hydrochloric acid solution (9.0 μl)
Was added to neutralize. Acetonitrile (100 μl) and sterile water (335 μl) were added to the neutralized reaction solution (65 μl).
Was added to bring the total volume to 500 μl. The solution was centrifuged (11000 rpm, 28 minutes) using Microcon Filter YM10 (Millipore), and 20% acetonitrile solution (100 μl) was further added and centrifuged (11000 rpm, 13 minutes).
And washed. After repeating the same washing operation, the solution remaining on the filter was recovered by reverse centrifugation and further 20%
After washing with an acetonitrile solution (25 μl), it was collected. The solvent of the obtained solution was distilled off under reduced pressure, and the solution was dissolved in 100 μl of sterilized water. Fluorescein or BHHCT-conjugated 18-base oligonucleotide was added to dATP, dGTP, dTTP, biotin
Reverse transcription was performed in the presence of -dCTP. The strand was directly immobilized on a streptavidin-coated plate.
In Fig. 4a, X is fluorescein or BHHCT
Indicates.

Example 7 Fluorescence measurement From 100 μl of the solution after the reverse transcription reaction obtained in Example 6, 1 μl, 2 μl, 4 μl or 10 μl was taken, and PBS solution (x10, 100 mM sodium phosphate pH 7.4, 1.4 M sodium chloride: 5 μl) was used. ) And sterile water for a total volume of 50 μl
Was prepared. Solution containing the above cDNA (50 μl)
Was added to a 96-well immunoplate on which streptavidin had been immobilized, and the mixture was allowed to stand at 4 ° C for 12 hours. After collecting the solution on the plate, PBST solution (10 mM sodium phosphate pH7.4, 140 mM sodium chloride, 0.05% Tween-2
0: 200 μl) 3 times, washing buffer solution (50 mM Tris-HCl pH 7.5, 50 mM sodium chloride: 200 μl)
The plates were washed twice with. Then, a fluorescence measurement buffer solution (2 μM europium chloride, 50 mM Tris-hydrochloric acid p
H7.5, 50 mM sodium chloride: 50 μl) was added, and fluorescence was measured. The fluorescence containing the cDNA derived from BHHCT-conjugated oligodeoxyribonucleotide (1) or fluorescein-conjugated oligodeoxyribonucleotide (2) was measured under the same conditions as in Example 5 using a multi-label counter (Wallac1420 ARVOsx, Wallac Berthold).

Polyribonucleotide (5) (100 pmol)
Fig. 4b shows the results obtained by adding 1 to 10% of the reverse transcription reaction from Escherichia coli to an immunoplate and measuring the fluorescence in the presence of europium ions. The vertical axis of the graph shows the value obtained by dividing each fluorescence intensity value by the background value. As a comparative experiment, a reverse transcription reaction was performed using dCTP instead of biotin-dCTP. After measuring the fluorescence of each reaction, EDTA solution (50 mM
After washing with EDTA-Na ₂ and 50 mM Tris-HCl (pH 7.5), fluorescence was measured again. Each result is shown as EDTA.

As a result of fluorescence measurement, the fluorescence derived from fluorescein-bound oligodeoxyribonucleotide (2) showed a small increase in fluorescence intensity with the increase in the amount of cDNA added to the plate, whereas the BHHCT-bound oligodeoxyribonucleotide ( The cDNA derived from 1) greatly increased depending on the amount of cDNA on the plate. The fluorescence intensity of BHHCT was more sensitive to the change in concentration than the fluorescence intensity of fluorescein, and thus it was clarified that it is more suitable for quantification of the amount of cDNA obtained by the reverse transcription reaction, that is, RNA. In addition, since the reverse transcription reaction product containing no biotin-bound dCTP, which was used as a comparative experiment, was not immobilized on the streptavidin plate, no fluorescence was observed. Therefore, biotin-d
The fluorescence observed in the presence of CTP was
It is the fluorescence derived from the cDNA in which n-dCTP was incorporated, and not the primer. When BHHCT-conjugated oligodeoxyribonucleotide (1) was added to the immunoplate for comparison, the primer alone was also adsorbed on the plate. However, 0.1% BS
When BHHCT-conjugated oligodeoxyribonucleotide (1) was added in the presence of A, adsorption to the plate was suppressed.

Example 8 Labeling of cDNA after Reverse Transcription with BHHCT The cDNA obtained after the reverse transcription reaction from RNA was labeled with BHHCT and the fluorescence was measured. The outline of the experiment is shown in FIG. 5a. Template RNA
As polyribonucleotide (5) (100 pmol, 0.
86 μl) was used as a primer, and oligodeoxyribonucleotide (1) or fluorochrome non-conjugated primer (3) (100 pmol, 2 μl) coupled with a terminal amino group was used as a reverse transcription reaction buffer solution (x5, 250 mM Tris-HCl pH8.3, 37).
The mixture was mixed with 5 mM KCl, 15 mM MgCl ₂ , 50 mM DTT: 2.0 μl) and sterilized water (0.64 μl), heated at 65 ° C. for 2 minutes, and then left at room temperature for 5 minutes. 10mM dATP, 1
0 mM dGTP and 10 mM dCTP (0.5 μl) were added respectively, and 5 mM dTTP (1 μl) or 5 mM 5- (3 ″ -aminopropynyl) -2′-deoxyurid was added.
ine 5'-triphosphate (dPTP; 1 μl) or 5 mM
5- [3 ''-aminoallyl] -2'-deoxyuridine-5'-triphos
phate (AA-dUTP; 1 μl), or 5 mM dTTP (0.
4 μl) and 5 mM dPTP (0.6 μl) or 5 mM dTTP
(0.4 μl) and 5 mM AA-dUTP (0.6 μl) were added,
Subsequently, M-MLV (200 units, 1 µl) was added, and the reaction was carried out at 37 ° C. 1 hour later M-MLV (10
0 units, 0.5 μl) was added, and the reaction was further performed at 37 ° C. for 1 hour. 200 mM EDTA-Na ₂ (10 μl), 5N
Sodium hydroxide (1.6 μl), sterile water (22.7
μl) was added and the mixture was heated at 65 ° C. for 30 minutes. After neutralization by adding 1N hydrochloric acid (7.2 μl), phenol extraction, phenol / chloroform extraction, and chloroform extraction were performed, and 3M ammonium acetate and ethanol were added to perform ethanol precipitation to obtain a cDNA chain. 0.5 for the obtained cDNA
Dissolve in 30 μl of M sodium carbonate buffer (pH 9.5),
BHHCT (1μmol / 60μl) dissolved in acetonitrile 2
0 μl was added and shaken at room temperature. About 12 hours later 10M
Ammonium acetate and ethanol were added and ethanol precipitation was performed to obtain cDNA with BHHCT bound thereto. After ethanol precipitation, the residue was dissolved in 100 μl of sterilized water.

Reference Example 2 Oligonucleotide (6) complementary to 3'end of cDNA obtained by reverse transcription reaction of polyribonucleotide (5)
Synthesis and purification of (SEQ ID NO: 4) were performed as follows. In order to bind biotin to the 5'end of the oligonucleotide (6), 3'was synthesized according to a usual synthesis method, and then 5'-biotin phosphoramidite (Glen Research) was finally bound. After completion of the synthesis, oligodeoxyribonucleotide (1) and oligodeoxyribonucleotide (3) were treated and purified as follows. The oligodeoxyribonucleotide was cut out from CPG (Controlled Pore Glass) with concentrated ammonia water and heated at 55 ° C. for 6 hours. After distilling off the solvent and dissolving in deionized water,
Purified by reverse phase HPLC.

The reverse phase HPLC conditions were as follows: Reverse phase HPLC column: μ-bonder sphere (C-18) column, Φ.
3.9 × 150 mm (manufactured by Waters) Solvent: A solution 5% Acetonitrile / 0.1M TEAA
(PH 7.0); B solution 50% acetonitrile / 0.1
M TEAA (pH 7.0).

Reversed phase HPLC of oligonucleotide (6)
Table 3 shows the conditions and the retention time of the linear concentration gradient in.

[0082]

[Table 3]

Example 9 BHHCT-labeled cDNA (10 μl) obtained in Example 8
And an oligonucleotide complementary to the 3'end of the cDNA (6)
(3 pmol), PBS (x10; 3 μl), 2% BSA (1.5
μl) and sterilized water were added to make the total volume 30 μl, and the mixture was heated at 90 ° C. for 2 minutes and then gradually cooled to bind the cDNA to the oligonucleotide (6). A solution (30 μl) containing the above cDNA was added to a 96-well immunoplate on which streptavidin was immobilized in advance, and the mixture was left at 4 ° C. for 12 hours. Since biotin is bound to the 5'end of the oligonucleotide (6), it is immobilized on a streptavidin plate. After collecting the solution on the plate, PBST solution (10 mM sodium phosphate p
H7.4, 140 mM sodium chloride, 0.05% Tween-20: 20
Washing buffer solution (50 mM Tris-HCl p
The plate was washed twice with H7.5, 50 mM sodium chloride: 200 μl). Then, a buffer solution for fluorescence measurement (2 μ
M europium chloride, 50 mM Tris-HCl pH 7.5, 50 m
M sodium chloride: 50 μl) was added and fluorescence was measured. Since fluorescein is bound to the 5'end of the oligonucleotide (2), the fluorescence of fluorescein can also be measured. Triphosphate added during reverse transcription is dA
TP, dCTP, dGTP, dTTP, but when dPTP or AA-dUTP is added instead of dTTP, dPTP or AA-dUTP is DNA
It is taken in and reacts with BHHCT. Further, the oligonucleotide (6) is an oligonucleotide that fixes and immobilizes DNA, and the labeled cDNA is immobilized only in the presence of the oligonucleotide (6). The result of the fluorescence measurement is shown in FIG. 5b.

Since strong fluorescence was observed specifically in the presence of the oligonucleotide (6) and dPTP or AA-dUTP, it was possible to label with BHHCT after the reverse transcription reaction and to show extremely high fluorescence intensity. Became clear. Especially in the reaction using AA-dUTP, a high labeling rate was obtained. In addition, in the reaction in which dTTP and dPTP or AA-dUTP coexist at a ratio of 2: 3, the fluorescence intensity is
Although it decreased due to the uptake of dTTP, it was revealed that labeling was possible even under such conditions. It has also been shown that the highly BHHCT-labeled cDNA obtained by such a post-reverse transcription modification method serves as a reagent for detecting complementary DNA or RNA.

Example 10 BHHCT-2′-deoxyuridine-
5'-triphosphate (BHHCT-2'-deoxyurydine-5'-triphospha
te (BH-dUTP)) (Fig. 6) (1) 5- (3 ''-Aminopropynyl) -2'-deoxyuridine 5'-tri
Phosphate (dPTP) was reported by Benner et al. (J. Am. Chem.
Soc., (1999), 121, 9781-9789) (FIG. 6A scheme I). (2) Reaction of BHHCT with 6-aminohexanoic acid (Fig. 6Bsc
heme II) BHHCT was synthesized according to the paper (Analytical Chemistry (1998), 70, 596-601).

170 mg (0.207 mmol) of BHHCT was dissolved in 3 ml of DMF, and 28 μl (0.2 μmo) of triethylamine was stirred therein.
l) and 236 mg (1.8 mmol) of 6-aminohexanoic acid are added,
The reaction started. After reacting at room temperature for 2 hours, ethyl acetate 30 m
l and 30 ml of 0.1 N hydrochloric acid aqueous solution were added for liquid separation. After repeating this extraction operation three times, the organic layers were collected and the solvent was distilled off.
The residue was purified on a silica gel column (the product was eluted with 1% methanol: chloroform containing a small amount of acetic acid),
The solvent was distilled off to obtain N-BHHCT- (6-aminohexanoic acid). Yield 150 mg (0.167 mmol), yield 81%. Mass spectrum result: FAB-HRMS theoretical value C ₃₆ H
₂₇ F ₁₄ NO ₈ S (M + H) +, 899.65, found 900.1259. Further
The structure of the product was confirmed by 1H-NMR.

(3) Active esterification of N-BHHCT- (6-aminohexanoic acid) N-BHHCT- (6-aminohexanoic acid) 140 mg obtained in Example 1
(0.16 mmol) was dissolved in 1.5 ml of DMF, and N-hydroxysuccinimide 36.8 mg (0.32 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 98.2 mg were dissolved.
(0.512 mmol) was added and the mixture was stirred at room temperature. After 30 hours, 30 ml of ethyl acetate and 30 ml of 0.1 N hydrochloric acid aqueous solution were added to separate the layers. After repeating this liquid separation operation, the aqueous layer was washed with ethyl acetate.
Back extraction was performed with ml, and the solvent in which the organic layer was collected was distilled off. The residue was purified on a silica gel column (the product was eluted with 1% methanol: chloroform containing a small amount of acetic acid) and the solvent was evaporated to give N-BHHCT- (6-aminohexanoic acid) succinimidyl ester. Obtained. Yield 50 mg (0.05 mmol), yield 31%. Mass spectrum result: FAB-HRMS theoretical value C ₄₀ H
₃₀ F ₁₄ N ₂ O ₁₀ S (M + H), 996.72, found 997.1495. Furthermore, the structure of the product was confirmed by 1H-NMR.

(4) BHHCT-2'-deoxyurydine-5'-triphos
Synthesis of phate (BH-dUTP) (Fig. 6 Cscheme III) dPTP (1.0 μmol) in 0.5 M sodium carbonate buffer (pH
9.5, 100 μl), 40 mM N-BHHCT- (6-aminohexanoic acid) succinimidyl ester acetonitrile solution (50 μl, 2.0 mmol) was added, and the mixture was stirred at room temperature to start the reaction. After 30 minutes, the above N-BHHCT- (6-aminohexanoic acid) succinimidyl ester acetonitrile solution (50 μl, 2.0 μmol) was further added. After 20 hours, acetonitrile was distilled off from the reaction solution under reduced pressure to remove deionized water (100 μm).
l), ethyl acetate (200 μl x 3) and add excess
N-BHHCT- (6-aminohexanoic acid) succinimidyl ester and reaction debris were removed. Place the aqueous phase on the reverse phase C8 column (Seppack back cartridge: YMC),
The column was eluted with a 50% aqueous acetonitrile solution. The eluted fraction was concentrated under reduced pressure, and the product was further purified by reverse phase HPLC. The collected liquid was concentrated under reduced pressure, and then desalted by the above-mentioned reverse phase C8 column. After elution from the column with 50% acetonitrile aqueous solution, the solvent was distilled off under reduced pressure, and BH-dUTP 12.4 OD ₃₃₀
units (from ε ₃₃₀ 3.41 x 10 ⁴ M ^-1 cm ^{-1 2} 36.2 nmol,
Yield 3.6%) was obtained.

The reverse phase HPLC conditions were as follows: Column: μ-Bondasphere (C-8) column Φ3.
9 × 150 mm (manufactured by Waters) Solvent: A solution 25% acetonitrile / 0.1M TEA
A (pH 7.0); B solution 50% acetonitrile / 0.
1M TEAA (pH 7.0).

[0090]

[Table 4]

Example 11 Oligodeoxyribonucleotide (7) used for enzymatic incorporation reaction of BH-dUTP (7)
Synthesis of oligodeoxyribonucleotide (7) was carried out in the same manner as in Example 1 using an automatic DNA synthesizer and deprotected.
Purified to high purity using reverse phase and ion exchange HPLC. Reversed phase and ion exchange HPLC conditions were as follows: Reversed phase HPLC column: μ-bonder sphere (C-18) column, Φ.
3.9 × 150 mm (manufactured by Waters) Solvent: A solution 5% Acetonitrile / 0.1M TEAA
(PH 7.0); B solution 25% acetonitrile / 0.1
M TEAA (pH 7.0).

Ion exchange HPLC column: TSKgel DEAE 2SW column, 4.6 × 250 mm,
Tosoh Co., Ltd. solvent: A solution 20% acetonitrile; B solution 20%
Table 5 shows the conditions and retention time of a linear concentration gradient of 2M HCOONH ₄ oligoribonucleotide (7) containing acetonitrile (SEQ ID NO: 5) in reverse phase and ion exchange HPLC.

[0093]

[Table 5]

Example 12 Synthesis and Purification of Template RNA and Oligoribonucleotide (8) Used for Uptake of BH-dUTP by Reverse Transcriptase Template RNA used for reverse transcription of BH-dUTP (oligoribonucleotide (8)) ( SEQ ID NO: 6) was synthesized as follows,
It was separated and purified. Also oligoribonucleotides (9)
(SEQ ID NO: 7) is a template having continuous adenine bases
It was RNA, and was synthesized in order to perform an experiment for examining whether or not BH-dUTP was continuously incorporated even in such a template RNA.

Using a ribonucleoside 3'-phosphoramidite (GLEN Research), an automatic DNA synthesizer (Applied
Oligoribonucleotides (8) and (9) were synthesized with Biosystem Model 394A). RNA fragment is 1 μmol
Synthesized on a scale. After completion of the synthesis, the synthesized oligonucleotide-bound CPG (Controlled Pore Glass) was treated with a mixed solution of concentrated aqueous ammonia: ethanol (3: 1 v / v) at room temperature for 2 hours and further heated at 55 ° C. for 16 hours. The solvent was distilled off, and 1 ml of 1M TBAF (Tetrabutylammonium) was added to the residue.
Fluoride) / THF (Tetrahydrofuran) solution is added, 37
The mixture was stirred at 0 ° C for 16 hours. Add 5 ml of 0.1M triethyl to it.
After adding ammonium acetate (pH 7.0), C18 silica gel (manufactured by Waters) column chromatography was performed (column size 1.5 × 12 cm: 5-40% acetonitrile, 50 mM Triethylammonium bicarbonate).
Elution with a concentration gradient using an aqueous solvent). About 30%
The fractions with the color development of dimethoxytrityl which were eluted with a concentration of acetonitrile were collected and 5 ml of 0.01 N
Hydrochloric acid was added and stirred for 1 hour. After neutralization with 0.1N ammonia water, the aqueous layer was washed with ethyl acetate, the solvent was distilled off, and the residue was dissolved in 1 ml of sterilized water. The oligoribonucleotides in this fraction were collected by reverse phase HPLC and then by ion exchange HPLC,
Purified. These oligonucleotides were subjected to the experiment described below in order to investigate the incorporation of BH-dUTP in the reverse transcription reaction. Oligonucleotides (8) and (9) were purified to high purity by reverse phase HPLC and ion exchange HPLC.

Reversed phase and ion exchange HPLC conditions were as follows: Reversed phase HPLC column: μ-Bondasphere (C-18) column, Φ.
3.9 × 150 mm (manufactured by Waters) Solvent: A solution 5% Acetonitrile / 0.1M TEAA
(PH 7.0); B solution 25% acetonitrile / 0.1
M TEAA (pH 7.0).

Ion exchange HPLC column: TSKgel DEAE 2SW column, 4.6 × 250 mm,
Tosoh Co., Ltd. solvent: A solution 20% acetonitrile; B solution 20%
Table 6 shows linear gradient conditions and retention times in reversed-phase and ion exchange HPLC of 2M HCOONH ₄ oligoribonucleotides (8) and (9) containing acetonitrile.

[0098]

[Table 6]

[0099]Reference example 3  5'-³²P-labeled oligonucleoti
Preparation of code (7) Oligoribonucleotide prepared as above (7)
[Γ- to (150 pmol) ³²P] ATP (3 μCi) 0.
3 μl, 5'end labeling reaction buffer (x10, 500 m
M Tris-hydrochloric acid (pH 8.0), 100 mM Magnesium chloride
Nesium, 50 mM dithiothreitol) 2 μl, T4
Polynucleotide kinase (10units); Takara Shuzo
(Manufactured by Co., Ltd.) 1 μl and sterilized water were added to give a total volume of 20 μl
And incubate at 37 ° C for 1 hour.
The 5'end of Cleotide (7) was labeled. NEN the reaction solution
Enzymes and salts were removed using SORB column (NEN)
After that, it was eluted with a 50% ethanol aqueous solution. Solvent under reduced pressure
After evaporation, 5'-labeled oligo dissolved in 400 μl of sterilized water
Nucleotide (7) was obtained.

Example 13 Reverse Transcription Reaction The oligodeoxyribonucleotide (7) (0.5 pmol) labeled with ³² P at the 5'end obtained in Reference Example 3 and the template oligoribonucleotide (8) (1 pmol) were added to dimethyl sulfol. Foxoxide (1 μl), 100 μM BH-dUTP (1 μl), 1
0 μM dNTP (dATP, dGTP or dCTP; 1 μl) was added to a reaction buffer solution (x5, 250 mM Tris-HCl pH 8.3, 375 m).
M potassium chloride, 15 mM magnesium chloride, 50 m
Mix with M dithiothreitol; 2 μl) and add M-MLV reverse transcriptase (10 units, 1 μl; Promega) to a total volume of 2
The reverse transcription reaction was performed with 0 μl at 37 ° C. The reaction solution (2.5 μl) was sampled after 10, 20 and 30 minutes, and the reaction stopping solution (50 mM EDTANa ₂ , 10 M urea,
After the reaction was stopped by adding 0.1% bromophenol blue (5 μl), 20% denaturing polyacrylamide gel electrophoresis containing 8 M urea was performed for analysis.

As a result of the reaction, a band migrating in the gel later than the mobility of oligodeoxyribonucleotide (7) was observed (FIG. 7). This indicates that BH-dUTP was incorporated by M-MLV reverse transcriptase at the 3'end of oligodeoxyribonucleotide (7). It was also shown that the elongation reaction did not occur in the absence of BH-dUTP, and that the elongation of the DNA chain occurred after the incorporation of BH-dUTP. It was also shown that the elongation reaction occurred similarly when oligoribonucleotide (9), which was RNA having a continuous adenine sequence, was used as a template.

Example 14 Terminal Transferase Oligodeoxyribonucleotide (7) (1 pmol) labeled with ³² P at the 5'end, BH-dUTP (510 pmol) was added to dimethyl sulfoxide (2 μl), 25 mM cobalt chloride solution ( 2 μl), reaction buffer solution (x5, 1 M potassium cacodylate, 25 mM Tris-hydrochloric acid, 1.25 mg / BSA, pH 6.6;
4μl), Terminal Transfer Race (25units)
Sterilized water was added to the mixture to make the total volume 20 μl, and the reaction was carried out at 37 ° C. 2, 4, 6, 8, 10, 12, 14 after starting the reaction
After 15 minutes, the reaction solution (2.5 μl) was sampled, added to a reaction stop solution (50 mM EDTANa ₂ , 10 M urea, 0.01% bromophenol blue; 5 μl) to stop the reaction, and then 15% modified polyacrylamide containing 8 M urea. Gel electrophoresis was performed and analyzed (FIG. 8).

As a result of the reaction, an oligonucleotide in which several BH-dUTPs were added to the 3'end of oligodeoxyribonucleotide (7) was observed, and it was revealed that BH-dUTP serves as a substrate for terminal transferase. . This showed that the 3'end of the oligonucleotide can be highly labeled with BHHCT.

[0104]

INDUSTRIAL APPLICABILITY The present invention has found an efficient method for synthesizing β-diketone-type europium complex-bonded nucleic acid and used them.
It enabled PCR, reverse transcription, and advanced labeling of cDNA. The labeled nucleic acid of the present invention has high sensitivity and is capable of detecting and quantifying a trace gene derived from a living body. Further, since the fluorescence peaks to be measured do not overlap with Cy3 and Cy5 which are generally used at present, they can be used in combination with these, and double and triple labeling can be performed. The detection of a target substance (nucleic acid) by fluorescence can be used, for example, for in situ hybridization, and is also suitable for quantifying a gene product immobilized on a stationary phase. Furthermore, in the currently produced DNA chips (DNA microarrays), the amount of DNA immobilized on the glass stationary phase has not been accurately quantified, so that the product has not been accurately evaluated. However, by immobilizing the PCR product or the synthetic gene with the β-diketone-type europium complex by the method according to the present invention and then immobilizing it on the substrate, it becomes possible to quantify the immobilized DNA,
It can be used for inspection before shipping the DNA chip, and more accurate supply of the DNA chip becomes possible. Furthermore, cd
Since a method for highly labeling NA with a β-diketone-type europium complex was found, it was also shown that a very small amount of change in gene expression that could not be detected by conventional fluorescent dyes could be detected by a chip.

[0105]

[Sequence list]                                SEQUENCE LISTING         <110> DNA Chip Research Inc.       Hitachi Software Engineering Co., Ltd.       Kazuko Matsumoto <120> Labeled Nucleic Acid and Method for Producing the same <130> 12B064 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 ctagccatat gtatatct 18 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 caaggagatg gcgcccaaca gtccccc 27 <210> 3 <211> 79 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic RNA <400> 3 ggggaauugu gagcggauaa caauuccccu cuagaaauaa uuuuguuuaa cuuuaagaag 60 gagauauaca uauggcuag 79 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 ggggaattgt gagcggataa caat 24 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 gttttcccag tcacgac 17 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic RNA <400> 6 cguagucgug acugggaaaa c 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic RNA <400> 7 gcaagucgug acugggaaaa c 21

[0106]

[Sequence list free text]

SEQ ID NO: 1 synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic RNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic RNA SEQ ID NO: 7: synthetic RNA

[Brief description of drawings]

FIG. 1 shows the structure of BHHCT.

FIG. 2 shows the binding of BHHCT to the ends of the primer and the structure of the fluorescein-bound primer.

FIG. 3 shows the results of fluorescence measurement of PCR products using fluorescein or BHHCT-binding primers.

FIG. 4 shows a schematic diagram (a) of a reverse transcription reaction using a fluorescein- or BHHCT-binding primer and a result of fluorescence measurement (b).

FIG. 5: After reverse transcription reaction in the presence of dPTP or AA-dUTP,
A schematic diagram (a) in which the obtained cDNA is labeled with BHHCT and a result (b) of fluorescence measurement are shown. U is dPTP incorporated into the cDNA strand
Or it shows AA-dUTP, and BHHCT reacts randomly with some of them. BG is background value, AA is AA-dUT
P and P represent dPTP, respectively.

FIG. 6A shows Scheme I (dPTP synthesis) for the synthesis of BH-dUTP.

FIG. 6B shows Scheme II for the synthesis of BH-dUTP (synthesis of N-BHHCT- (6-aminohexanoic acid) succinimidyl ester).

FIG. 6C: Scheme III for the synthesis of BH-dUTP (Synthesis of BH-dUTP)
Indicates.

FIG. 7 shows the sequences used in the BH-dUTP incorporation experiment in the reverse transcription reaction and the result of autoradiography of the analysis of the reaction.

FIG. 8 shows the results of autoradiography of the reaction of addition of BH-dUTP to the 3 ′ end of oligonucleotide (7) by terminal transfection.

FIG. 9 shows an example of a method for quantifying an immobilized gene by immobilizing a PCR product or an oligonucleotide labeled with BHHCT on a stationary phase and then performing time-resolved fluorescence measurement in the presence of europium ions.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 G01N 33/566 33/543 575 33/58 A 33/566 C12N 15/00 ZNAA 33/58 F (72) Inventor Yasuo Komatsu 134 DK Kobe Co., Ltd., Hodogaya-ku, Yokohama, Kanagawa Pref. 72 Einko Otsuka 134 KOBE, Hodogaya-ku, Yokohama, Kanagawa DK Chip Lab. 72) Inventor Kazuko Matsumoto 3-9-12-105 F-term, Setagaya-ku, Tokyo 2F (reference) 2G045 AA35 DA12 DA13 DA14 FB02 FB07 FB12 GC15 4B024 AA11 AA20 CA06 CA09 HA08 HA12 HA14 4B029 AA07 AA23 FA12 4B063 Q42 QRAQ QR62 QR82 QS03 QS25 QS34 QX02

Claims (31)

[Claims] 1. A compound represented by the general formula (I): [In formula, n shows the integer of 1-6. ] The labeled compound represented by the following formula is represented by the following general formula (II): [In the formula, a represents an integer of 1 to 10, and X represents a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the base, phosphate, sugar moiety or nucleic acid of the nucleic acid. ] Or the general formula (III): [In the formula, b, d, and f are the same or different and represent an integer of 0 to 10, and c and e are the same or different and are 0.
Alternatively, it represents an integer of 1. However, b, d, and f are all 0
Not included if X represents a base of a nucleic acid, a phosphate, a sugar moiety or a saturated or unsaturated hydrocarbon having 6 or less carbon atoms bonded to the nucleic acid. ] The labeled nucleic acid couple | bonded with the nucleic acid through the linking group which has the amino group shown by these. 2. A compound represented by the general formula (I): [In formula, n shows the integer of 1-6. ] The labeled compound represented by the following formula is represented by the following general formula (IV): [Wherein R 1, R 2 and R 3 are the same or different and each represents a hydrogen atom, or an aminoalkyl group represented by the above general formula (II) or (III), and Y represents a base of a nucleic acid, a phosphate, or a sugar moiety. Represent ] The labeled nucleic acid couple | bonded with the nucleic acid through the coupling group shown by these. 3. A compound represented by the general formula (I): [In formula, n shows the integer of 1-6. ] The labeled compound represented by the following is a labeled nucleic acid bound to a nucleic acid through a linking group composed of aminoalkyl, and has the general formula (V): [In the formula, N represents either a 2'-deoxythymidine nucleotide, a 2'-deoxyadenine nucleotide, a 2'-deoxycytosine nucleotide or a 2'-deoxyguanine nucleotide, n is an integer of 1 to 6, and i is 1 The integer of -10, q represents the integer of 1-100. ] The said labeled nucleic acid shown by these. 4. A compound represented by the general formula (I): [In formula, n shows the integer of 1-6. ] The labeled compound represented by the following is a labeled nucleic acid bound to a nucleic acid through a linking group consisting of aminoalkyl, and has the general formula (V): [In the formula, N represents any one of a uridine nucleotide, an adenine nucleotide, a cytosine nucleotide, or a guanine nucleotide, n is an integer of 1 to 6, and i is 1 to 10
, And q represents an integer of 1 to 100. ],
The labeled nucleic acid. 5. A compound represented by the general formula (I): [In formula, n shows the integer of 1-6. ] The manufacturing method of the labeled nucleic acid of any one of Claim 1 to 4 which couple | bonds the labeled body shown by these with a nucleic acid through a coupling group. 6. A method for amplifying a nucleic acid, which uses the labeled nucleic acid according to claim 1 as a probe. 7. A method for isolating a reaction product by amplifying the nucleic acid using the labeled nucleic acid according to any one of claims 1 to 4 as a probe and then removing excess primer from the reaction product. 8. A reverse transcription reaction using the labeled nucleic acid according to any one of claims 1 to 4 as a primer. 9. A reaction product obtained by the reverse transcription reaction according to claim 8. 10. A fluorescent complex obtained by adding a heavy metal ion to the labeled nucleic acid according to claim 1 and reacting with a target substance on a biological sample, and measuring the fluorescence of the fluorescent complex after the reaction. And a method for analyzing nucleic acid. 11. A method of reacting the labeled nucleic acid according to claim 1 with a target substance on a biological sample, adding a heavy metal ion, and measuring the fluorescence of the resulting fluorescent complex. And a method for analyzing nucleic acid. 12. A fluorescent complex containing the labeled nucleic acid according to claim 1 and a heavy metal ion. 13. The fluorescent complex according to claim 12, wherein the heavy metal ion is a lanthanoid metal ion or a radium ion. 14. The fluorescent complex according to claim 13, wherein the lanthanoid metal ion is an ion of europium, samarium, terbium or dysprosium or a mixture thereof. 15. The labeled nucleic acid according to claim 1, which is reversibly or irreversibly immobilized on a stationary phase. 16. A method for quantifying a nucleic acid on a stationary phase by adding a heavy metal ion to the labeled nucleic acid according to claim 15 and measuring the fluorescence of the resulting fluorescent complex. 17. A method of adding a heavy metal ion to the labeled nucleic acid according to claim 1, immobilizing it reversibly or irreversibly on a stationary phase, and measuring the fluorescence of the resulting fluorescent complex. The method for quantifying nucleic acids on a stationary phase according to. 18. A compound represented by the general formula (VI): Or general formula (VII) [Wherein R ₁ and R ₂ are each a 2′-deoxythymidine nucleotide, a 2′-deoxyadenine nucleotide, a 2′-deoxycytosine nucleotide, a 2′-deoxyguanine nucleotide, a general formula (VI) or a general formula (VII). Label-bound 2'-deoxyoligoribonucleotide consisting of 2'-deoxyuridine derivative, n is an integer of 1 to 6, and h and m are the same or different and are an integer of 1 to 6. ] The 2'-deoxy polyribonucleotide which has the label | marker coupling | bonding 2'-deoxy uridine derivative shown by these in at least 1 residue chain. 19. The following general formula (VIII): Or the following general formula (IX): [H and m are the same or different and each represents an integer of 1 to 6. ] A deoxypolyribonucleotide containing at least one compound represented by the formula: [In formula, n shows the integer of 1-6. ] The method for producing a 2'-deoxypolyribonucleotide according to claim 18, which comprises binding the label shown in []. 20. The reaction of the 2′-deoxypolyribonucleotide according to claim 18 with a target substance on a biological sample, followed by adding a heavy metal ion, and measuring the fluorescence of the resulting fluorescent complex. Analytical method for target substances. 21. The fluorescence of the resulting fluorescent complex, which is obtained by reacting a target substance on a biological sample after adding a heavy metal ion to the 2'-deoxypolyribonucleotide according to claim 18, is measured. Analytical method for target substances. 22. The general formula (X): [In the formula, h is 1 to 6, e is 1 to 10, and n is an integer of 1 to 6. ] 2′-deoxyuridine-
5'-triphosphoric acid. 23. The label-bound 2′-deoxyuridine-5′-triphosphate according to claim 22, wherein e is 5, h is 1 and n is 3. 24. (1) General formula (XI): [In formula, e represents the integer of 1-10. ] To the aminocarboxylic acid derivative represented by the general formula (I) [In formula, n shows the integer of 1-6. ] The labeled compound represented by the formula [XII] [Wherein, e is the same as the above], and a labeled body-bound aminocarboxylic acid body is obtained. (2) Next, the labeled body-bound aminocarboxylic acid body is treated with succinimide and 1- (3-dimethylaminopropyl). By reacting in the presence of -3-ethylcarbodiimide hydrochloride to obtain formula (XIII), (3) Next, the compound represented by the general formula (VIII): [H represents an integer of 1 to 6] ] The method of manufacturing the label | marker coupling | bonding 2'-deoxyuridine-5'-triphosphoric acid of Claim 22 by performing a condensation reaction with the compound shown by these. 25. A compound represented by the general formula (XIV): [In the formula, m represents 1 to 6, e represents 1 to 10, and n represents an integer of 1 to 6. ] 2′-deoxyuridine-
5'-triphosphoric acid. 26. The label-bound 2′-deoxyuridine-5′-triphosphate according to claim 25, wherein e is 5, m is 1, and n is 3. 27. A compound represented by the general formula (XIII): [In formula, e represents 1-10, n represents the integer of 1-6. ] The compound represented by the general formula (IX) [M represents an integer of 1 to 6] ] The labeled substance binding according to claim 25, characterized by reacting with a compound represented by
A method for producing 2'-deoxyuridine-5'-triphosphoric acid. 28. A method for labeling a nucleic acid using the label-bound 2′-deoxyuridine-5′-triphosphate according to claim 22 or 25. 29. A labeled nucleic acid obtained by the method according to claim 28. 30. After reacting the labeled nucleic acid according to claim 29 with a target substance on a biological sample, a heavy metal ion is added,
A method for analyzing a target substance, which comprises measuring the fluorescence of the generated fluorescent complex. 31. A heavy metal ion is added to the labeled nucleic acid according to claim 29, which is then reacted with a target substance on a biological sample.
A method for analyzing a target substance, which comprises measuring the fluorescence of the generated fluorescent complex.