JP2002355084A - New nucleic acid probe and new method for assaying nucleic acid using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a new nucleic acid probe for simultaneously assaying target nucleic acids with a simple apparatus when one more kinds of the target nucleic acids are present in the assaying system and a new method for assaying the nucleic acids using the probe. SOLUTION: This new nucleic acid probe for assaying the nucleic acids is characterized in that a base sequence is designed so as to reduce fluorescence intensities of both a donor dye and an acceptor dye when the probe is hybridized to the target nucleic acids and the dye is labeled in the single-stranded nucleic acid probe labeled with a plurality of fluorescent dyes so as to form at least one pair of fluorescent days causing a Forster resonance energy transfer (FRET) phenomenon, i.e., the pair of the fluorescent dye (the donor dye) convertible into the donor dye and the fluorescent dye (the acceptor dye) convertible into the acceptor dye. The new method for assaying the nucleic acids uses the probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a target nucleic acid present in a measurement system using a simple device when at least one or more (ie, one or more) target nucleic acids are present in the measurement system (a plurality of target nucleic acids). A new nucleic acid probe for simultaneously measuring the presence of a target nucleic acid, and a novel nucleic acid measurement method using the probe.

More specifically, Forster (or fluorescence) re
A fluorescent dye pair that causes the phenomenon of sonance energy transfer (hereinafter referred to as FRET), that is, a pair of a fluorescent dye that can be a donor dye (hereinafter referred to as a donor dye) and a fluorescent dye that can be an acceptor dye (hereinafter referred to as an acceptor dye). A novel nucleic acid probe for measuring single-stranded nucleic acid labeled with a plurality of fluorescent dyes so as to form at least one pair, a nucleic acid measuring method using the nucleic acid probe, a nucleic acid measuring reagent kit used in the method, and a target nucleic acid Polymorphism or / and mutation (mu)
The present invention relates to a method for analyzing or measuring tation) and a measurement kit used for the method.

[0003]

2. Description of the Related Art A nucleic acid probe in which a fluorescent dye pair causing a FRET phenomenon, that is, a donor dye fluorescein and an acceptor dye X-rhodamine, are linked to a single-stranded oligonucleotide is known (Experimental Medicine, vol. 15, vol. (No. 7, extra number), 728-733, 1
997). However, this nucleic acid probe is used for monitoring a PCR method, and is not intended to directly measure a nucleic acid.

In some cases, it has been used for the detection of target nucleic acids (Bi
ochemistry, 34, 285-292, 1995). In this case, when the nucleic acid probe is not hybridized to the target nucleic acid, the fluorescence intensity of the nucleic acid probe itself is at a low level due to the FRET phenomenon. That is, the emission of fluorescein is suppressed, and the fluorescence intensity is at a low level. On the other hand, the fluorescence intensity of X-rhodamine is high, but not as high as the suppressed emission of fluorescein. However, when the nucleic acid probe hybridizes to the target nucleic acid, the three-dimensional structure of the probe changes, and the FRET phenomenon is eliminated. As a result, the emission of fluorescein increases, and the fluorescence intensity of the reaction system increases. However, if the three-dimensional structure of the nucleic acid probe does not change after hybridization, no change occurs in the fluorescence intensity, and the fluorescence intensity is not suitable for nucleic acid measurement.

[0005] The present inventors have developed a nucleic acid probe which is a fluorescence quenching probe that reduces fluorescence after hybridization when hybridized to a target nucleic acid without the intervention of another nucleic acid probe (Nucleic Acid, 29).
No. 6e34, 2001; EP1046 717 A9; JP-A-2001-2863
00 (P2001-286300A). This nucleic acid probe is labeled with a fluorescent dye at the end of a single-stranded oligonucleotide,
Fluorescent dyes and base sequences are designed so that when they hybridize to a target nucleic acid, the fluorescence intensity decreases. This nucleic acid probe can accurately and easily measure a target nucleic acid in a minute amount. However, the present conventional method requires that G (guanine) and C (cytosine)
Fluorescence quenching (quenching due to the transfer of luminescence energy to the complex) due to the interaction between the base pair complex (hydrogen bond) and the fluorescent dye Since the amount of fluorescent light is very small, usable fluorescent emission colors are limited.

When many types of target nucleic acids are present in a measurement system, simultaneous measurement of all of them requires the same number of fluorescent colors as the target nucleic acids. In other words, it requires the same number of nucleic acid probes having different fluorescent emission colors as the target nucleic acid species. In this conventional method, in order to increase the fluorescent emission color, it is necessary to increase the number of fluorescent dyes having different fluorescence emission colors due to the interaction between the hydrogen bond of GC and the fluorescent dye. Because of the limited emission color, there has been a limit in detecting multiple (especially three or more) target nucleic acid species simultaneously. Furthermore, different fluorescent dyes mean different excitation wavelengths, which means that a large number of excitation light sources are required to excite all of these fluorescent dyes, and the device becomes large-scale. Was uneconomical.

[0007]

SUMMARY OF THE INVENTION In view of the above-mentioned circumstances, an object of the present invention is to provide a fluorescent dye pair that causes a FRET phenomenon to a single-stranded oligonucleotide, that is, a nucleic acid probe in which a donor dye and an acceptor dye are bonded to each other. A novel nucleic acid probe for nucleic acid measurement (hereinafter referred to as a novel nucleic acid measurement) capable of simultaneously measuring at least one or more (i.e., one or more) target nucleic acids with a measurement device and capable of measuring a small amount, in a short time, simply, and accurately. The nucleic acid probe may be simply referred to as a nucleic acid probe.) It is still another object of the present invention to provide a novel nucleic acid measurement method using the same (hereinafter, sometimes simply referred to as a nucleic acid measurement method), a reagent kit thereof, a method for measuring a target nucleic acid polymorphism / mutation, and a measurement kit thereof. .

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors, based on the fluorescence quenching probe,
By means of trial and error, various nucleic acid probes prepared by labeling a single-stranded oligonucleotide with various fluorescent dyes at two positions of the oligonucleotide were compared in detail. As a result, when a specific position of an oligonucleotide is labeled with a specific donor dye and an acceptor dye that is paired with the specific dye, the nucleic acid probe hybridizes to the target nucleic acid. Was hybridized to the target nucleic acid), the fluorescence intensity of the acceptor dye was significantly reduced, or the fluorescence intensity of both the donor and the acceptor dye was significantly reduced. The present invention has been completed based on such findings.

That is, the present invention provides: A fluorescent dye pair that causes the FRET phenomenon, that is, a fluorescent dye that can be a donor dye (hereinafter, referred to as a donor dye) and a fluorescent dye that can be an acceptor dye (hereinafter, referred to as a donor dye)
It is called an acceptor dye. In a single-stranded nucleic acid probe labeled with a plurality of fluorescent dyes so that at least one pair of the pair is formed, when the probe hybridizes to a target nucleic acid, the fluorescence intensity of the acceptor dye is reduced. 1. a nucleic acid probe whose base sequence is designed and the dye is labeled; The nucleic acid probe according to 1, wherein the fluorescence intensity of the donor dye and the acceptor dye both decreases,

[0010] 3. BODI is a fluorescent dye that can be a donor dye
PY FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamin
3. The nucleic acid probe according to the above 1 or 2, which is 6-TAMRA, or e. The nucleic acid probe according to any one of 1 to 3, which has a pair of dye pairs of a donor dye and an acceptor dye,

5. The nucleic acid probe is labeled at its end with a donor dye, and when the nucleic acid probe hybridizes to the target nucleic acid at the end, 1 to 3 bases away from the terminal base of the target nucleic acid hybridized to the probe. 5. The nucleic acid probe according to any one of 1 to 4, wherein the base sequence of the probe is designed such that at least one base of G (guanine) is present in the base sequence of the target nucleic acid; When the nucleic acid probe hybridizes to the target nucleic acid, the bases of the probe-nucleic acid hybrid form at least one pair of G (guanine) and C (cytosine) in the donor dye-labeled portion so as to form at least one pair of G (guanine) and C (cytosine). The nucleic acid probe according to any one of the above 1 to 5, wherein the sequence is designed,

7. 7. The nucleic acid probe according to any one of 1 to 6 above, wherein the donor dye labels the 5 ′ end (including the 5 ′ end) of the nucleic acid probe; 8. The nucleic acid probe according to any one of 1 to 7 above, wherein the donor dye labels the 3 ′ end (including the 3 ′ end) of the nucleic acid probe; 9. The nucleic acid probe according to the above item 7, wherein the 5 'terminal base of the nucleic acid probe is G or C and the 5' terminal is labeled with a donor dye. The nucleic acid probe according to the above 8, wherein the 3 ′ terminal base of the nucleic acid probe is G or C, and the 3 ′ terminal is labeled with a donor dye,

11. The nucleic acid probe according to at least one of the above items 1 to 10, wherein the nucleic acid probe hybridizes to the target nucleic acid, and a type of the target nucleic acid (hereinafter, referred to as a nucleic acid probe). The number of species is at least as many as the number of species, and nucleic acid probes having different emission fluorescent colors are present, and the nucleic acid probe and the target nucleic acid are hybridized to form a type of the nucleic acid probe (hereinafter, also simply referred to as a species). )), A method for measuring a decrease in fluorescence intensity at a measurement wavelength before and after hybridization with at least the same number of types of wavelengths (hereinafter, also simply referred to as species). 12. A kit for measuring the concentration of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present, wherein the kit for nucleic acid measurement contains or accompanies the nucleic acid probe according to at least one of the above 1 to 10. ,Also,

13. Target nucleic acid polymorphism in a measurement system in which at least one or more target nucleic acids are present
And / or a method for analyzing or measuring mutation, wherein the nucleic acid probe according to at least one of the above items 1 to 10, wherein the number of species is at least the same as the number of species of the target nucleic acid, and the luminescence is luminescence. Nucleic acid probes with different fluorescent colors are present in the measurement system, the target nucleic acid is hybridized with the nucleic acid probe, and the fluorescence intensity at the measurement wavelength before and after hybridization is at least as many as the number of species of the nucleic acid probe. 13. a method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, which comprises measuring the amount of decrease of the target nucleic acid; A measurement kit for analyzing or measuring a polymorphism or / and a mutation of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present, wherein the measurement kit contains or accompanies the nucleic acid probe according to at least one of the above 1 to 10. A measurement kit for analyzing or measuring a polymorphism or / and mutation of a target nucleic acid characterized by:

15. 14. The nucleic acid measuring method according to the above item 11 or 13, wherein the target nucleic acid is derived from a microorganism or animal obtained by pure separation. 16. 14. The nucleic acid measuring method according to the above item 11 or 13, wherein the target nucleic acid is a nucleic acid contained in a cell of a complex microbial system or a symbiotic microbial system or in a homogenate of the cell.

[0016]

Next, the present invention will be described in more detail with reference to preferred embodiments. The first invention of the present invention is the FR
When a single-stranded nucleic acid probe labeled with a plurality of fluorescent dyes so as to form at least one pair of a fluorescent dye pair that causes an ET phenomenon, i.e., a pair of a donor dye and an acceptor dye, when the probe hybridizes to a target nucleic acid In addition, the nucleic acid probe is characterized in that the fluorescent dye is labeled and the base sequence is designed so that the fluorescent intensity of the acceptor dye of the probe-nucleic acid hybrid complex decreases. Among them, preferred nucleic acid probes are those designed so that the fluorescence intensity of both the donor and acceptor dyes is reduced.

In the present invention, the probe-nucleic acid hybrid complex means a complex (complex) in which the nucleic acid probe of the present invention is hybridized with a target nucleic acid.
For the sake of simplicity, hereinafter, it is abbreviated as a nucleic acid hybrid complex.

In the present invention, DNA, RNA,
cDNA, mRNA, rRNA, XTPs, dXTP
s, NTPs, dNTPs, nucleic acid probe, helper nucleic acid probe (or nucleic acid helper probe, or simply helper probe), hybridization, hybridization, intercalator, primer, annealing, extension reaction, heat denaturation reaction, nucleic acid melting curve, PCR,
RT-PCR, RNA-primed PCR, Stretch PC
R, reverse PCR, PCR using Alu sequence, multiplex PC
R, PCR using mixed primers, P using PNA
CR method, hybridization assay method (hybrid
), FISH (fluorescent in situ hybri)
dization assays), PCR (polymerase chain)
assays), LCR method (ligase chain reaction), S
D method (strand displacement assays), competitive hybridization method, D
NA chip, nucleic acid detection (gene detection) device, S
Terms such as NP (snip: single nucleotide substitution polymorphism) and complex microbial systems have the same meanings as those generally used in molecular biology, genetic engineering, microbial engineering and the like at present.

In the present invention, nucleic acid measurement means quantifying or quantitatively detecting a target nucleic acid, or simply detecting it. In the present invention, a target nucleic acid refers to a nucleic acid or a gene for the purpose of measuring a nucleic acid. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids other than the target nucleic acid may be mixed. Then, at least one or more target nucleic acids exist in the measurement system. For example, a complex microbial system (R of multiple microorganisms)
A mixed system of NA or genetic DNA) or a symbiotic microbial system (RNA of multiple animals and plants and / or multiple microorganisms)
Or at least one specific nucleic acid for the purpose of measurement in a mixed system of gene DNA). If the target nucleic acid needs to be purified, it can be performed by a conventionally known method. For example, it can be performed using a commercially available purification kit or the like. As a specific example of the above nucleic acid, DN
A, RNA, PNA, oligodeoxyribonucleotides, oligoribonucleotides, etc., and chemically modified nucleic acids of the above nucleic acids can be mentioned. 2′- O -methyl (Me) RNA and the like can be exemplified as the chemically modified nucleic acid.

The donor dye which can be a donor dye in the FRET phenomenon in the present invention includes at least a. Is excited at a specific wavelength and emits at a specific wavelength; b. Can transfer luminescence energy to a specific dye (a dye that can be an acceptor dye), c. A complex of GC base pairs (hydrogen bond of GC base pairs) generated when the nucleic acid probe hybridizes with the target nucleic acid (hereinafter sometimes referred to as GC hydrogen bond for convenience), which is present near the donor dye In this case, it is defined as satisfying such a condition that energy can be transferred to the base pair. That is, any dye may be used as long as it satisfies this condition. Generally, a dye that can be a donor dye in the FRET phenomenon and that reduces the fluorescence intensity of the probe-nucleic acid hybrid complex when a nucleic acid probe that is itself labeled alone hybridizes to a target nucleic acid is preferably used (Nucleic acid). Acid, Vol. 29, No. 6e34, 2001
Year). For example, as the donor dye, BODIPY FL (trade name; manufactured by Molecular Probes, USA), BODIPY 493/503 (trade name; molecular
Probe (Molecular Probes, USA), 5-FAM, Te
tramethylrhodamine, 6-TAMRA, fluorescein (fluor
escein) or derivatives thereof (eg, fluorescein isothiocyanate (FI)
TC) or its derivatives, such as Alexa 488, Alexa 532, c
y3, cy5, EDANS (5- (2'-aminoethyl) amino-1-naphthalen
esulfonic acid)), rhodamine 6G (R6G) or a derivative thereof (eg, tetramethylrhodamine isothiocyanate).
e) (TMRITC), BODIPY FL / C3 (trade name; manufactured by Molecular Probes, USA), BODIPY FL / C6 (trade name; manufactured by Molecular Probes) , United States), BODIPY
5-FAM (trade name; Molecular Probe)
obes), USA), BODIPYTMR (trade name; Molecular Probes, USA), or a derivative thereof (eg, BODIPY TR)
(Trade name: Molecular Probes)
BODIPY R6G (trade name; Molecular Probes, USA),
BODIPY 564 (trade name; manufactured by Molecular Probes, USA), BODIPY
581/591 (trade name; Molecular Probe)
Probes), USA).

Among the above-mentioned preferred donor dyes, BO
DIPY FL (trade name; Molecular Probe)
Probes), BODIPY FL-based dyes (trade name; Molecular Probes, USA), BODIPY 493/503 (trade name; Molecular
Probe (Molecular Probes, USA), 5-FAM, BODIPY 5-FAM (trade name; molecular probe)
(Molecular Probes), USA), Tetramethylrhodami
ne, 6-TAMRA, etc., as BODIPY FL
(Trade name: Molecular Probe
s), USA), BODIPY 493/503 (trade name; Molecular Probes, USA), 5-FA
M, Tetramethylrhodamine, 6-TAMRA and the like can be mentioned. However, the present invention is not limited to these examples.

In the FRET phenomenon, an acceptor dye generally undergoes energy transfer from a donor dye, ie, a dye capable of becoming an acceptor dye in a pair with a donor dye (in other words, quenching (quenching action to a donor dye) Any dye can be used as long as the dye has a function. And it depends on the kind of donor dye forming a pair. For example, BODIPY FL
(Trade name: Molecular Probe
s), BODIPY FL-based dyes (trade name; Molecular Probes, USA), BODIPY 493/503 (trade name; Molecular Probes, USA) ), 5-FAM, BODIPY 5-FAM (trade name; manufactured by Molecular Probes, USA), Tetramethylrhoda
If mine, 6-TAMRA, or the like is used as the donor dye, X-rhodamine, BODIPY 581/591 (trade name, manufactured by Molecular Probes, USA), or the like can be used as the acceptor dye. . However, the present invention is not limited to these examples.

The nucleic acid probe of the present invention that hybridizes to a target nucleic acid may be composed of oligodeoxyribonucleotides or oligoribonucleotides. Alternatively, a chimeric oligonucleodite containing both of them may be used. These oligonucleotides may be chemically modified. The chemically modified oligonucleotide may be interposed in the chimeric oligonucleotide chain.

The modified sites of the chemically modified oligonucleotide include a terminal hydroxyl group or a terminal phosphate group at the terminal of the oligonucleotide, an internucleoside phosphate site, a carbon at the 5-position of the pyrimidine ring, and a nucleoside sugar (ribose). Or deoxyribose) site. Preferably, a ribose or deoxyribose site can be mentioned. Specifically, 2'- O-
Alkyl oligoribonucleotide (2'- O- alkyloligoribo
Nucleotides) (hereinafter, 2'- O -. the abbreviated to 2-O-), 2-
O-alkylene oligoribonucleotide (2-O-alkyleneol
igoribonucleotides) and 2-O-benzyloligoribonucleotides. The oligonucleotide is one in which the OH group at the 2′-position carbon of one or more riboses at any position of the oligoribonucleotide is modified (by an ether bond) with an alkyl group, an alkylene group or a benzyl group. In the present invention, preferably, among the 2-O-alkyl oligoribonucleotides, 2-O-methyl oligoribonucleotide, 2-O-ethyl oligoribonucleotide, 2-O-butyl oligoribonucleotide, O-benzyl oligoribonucleotides, among the 2-O-alkylene oligoribonucleotides, 2-O-ethylene oligoribonucleotides, and 2-O-benzyl oligoribonucleotides, particularly preferably 2-O- Methyl oligoribonucleotide (hereinafter simply referred to as 2-O-Me oligoribonucleotide) is used. By applying such a chemical modification to the oligonucleotide, the affinity for the target nucleic acid is increased, and the hybridization efficiency of the nucleic acid probe of the present invention is improved. When the hybridization efficiency is increased, the reduction rate of the fluorescence intensity of the fluorescent dye of the nucleic acid probe of the present invention is further improved, so that the measurement accuracy of the concentration of the target nucleic acid is further improved. In the present invention, the term “oligonucleotide” means oligodeoxyribonucleotide or oligoribonucleotide or both, and is generically referred to. 2-O-alkyl oligoribonucleotides, 2-O-alkylene oligoribonucleotides and 2-O-benzyl oligoribonucleotides can be prepared by known methods (Nucleic Acids Research, Vol. 26, 222).
4-2229, 1998). In addition, GENSET (Ltd.) (France) has commissioned synthesis, so it can be easily obtained. The present inventors commissioned the company to synthesize the compound and conducted experiments to complete the present invention.

Incidentally, 2-O-methyl oligoribonucleotide (hereinafter simply referred to as 2-O-Me oligoribonucleotide)
The nucleic acid probe of the present invention in which a modified RNA such as
When used for measurement of NA, particularly rRNA, preferable results can be obtained. When measuring RNA using the nucleic acid probe of the present invention, an RNA solution as a sample is subjected to 80 to 100 ° C., preferably 9 ° C. before hybridization with the probe.
Heat treatment at 0 to 100 ° C., optimally 93 to 97 ° C. for 1 to 15 minutes, preferably 2 to 10 minutes, optimally 3 to 7 minutes, destroys the higher order structure of RNA by hybridization. It is suitable for improving efficiency.

Further, in order to further increase the efficiency of hybridization of the nucleic acid probe of the present invention to a hybridization base sequence region, a helper probe (helper p) is used.
robe) is preferably added to the hybridization reaction solution. In this case, the oligonucleotide of the helper probe may be an oligodeoxyribonucleotide or an oligoribonucleotide, or may be an oligonucleotide subjected to the same chemical modification as described above. As an example of the oligonucleotide, (5 ′) TCCTTTGAGT TCCCGGCCG is used as a forward type in a PCR method.
GA (3 '), as backward or reverse ward (5') CCCTGGTCGT AAGGGCCA
TG ATGACTTGAC GT (3 '). Preferred examples of chemically modified oligonucleotides include 2-O-alkyl oligoribonucleotides,
In particular, 2-O-Me oligoribonucleotides can be exemplified.

When the nucleic acid probe of the present invention has a base chain of 35 bases or less, the use of a helper probe is particularly effective. When using the nucleic acid probe of the present invention having a length of more than 35 base chains, it may be sufficient to simply heat denature the target RNA. As described above, the nucleic acid probe of the present invention
When hybridized to NA, the hybridization efficiency increases, so that the fluorescence intensity decreases according to the amount of RNA in the reaction solution, and the final RNA concentration becomes approximately 150 pM.
Can be measured. Thus, the present invention provides a measurement kit for measuring the concentration of at least one or more (i.e., one or more) target nucleic acids containing or accompanying at least as many nucleic acid probes of the present invention as the number of target nucleic acid species. It is also a measurement kit containing at least one helper probe and measuring the concentration of at least one or more (ie, one or more) target nucleic acids.

In the measurement of RNA by a conventional hybridization method using a nucleic acid probe, an oligodeoxyribonucleotide or oligoribonucleotide has been used as a nucleic acid probe. Since RNA itself has a strong conformation, the probe and target R
The efficiency of hybridization with NA was poor, and the quantitativeness was poor. For this reason, the conventional method has the complexity of denaturing the RNA, fixing it to a membrane, and then performing a hybridization reaction. On the other hand, the method of the present invention uses a ribose moiety-modified nucleic acid probe having a high affinity for a specific structural part of RNA, so that the hybridization reaction can be performed at a higher temperature than the conventional method. . Therefore, the above-mentioned adverse effects of the higher-order structure of RNA can be overcome only by using a heat denaturation treatment and a helper probe as a pretreatment. Thereby, in the method of the present invention, the hybridization efficiency becomes substantially 100%, and the quantification is improved. In addition, it becomes much simpler than the conventional method.

The probe of the present invention has 5 to 60 bases, preferably 10 to 35, particularly preferably 15 to 20 bases.
It is. If it exceeds 60, the permeability of the cell membrane will be poor when used in the FISH method, and the application range of the present invention will be narrowed. If it is less than 5, non-specific hybridization is likely to occur, resulting in a large measurement error.

The structure is such that when a nucleic acid probe is labeled at its end with a donor dye and the nucleic acid probe hybridizes to the target nucleic acid at the end, the terminal base of the target nucleic acid hybridized to the probe is formed. 1 to 3 bases away from the base sequence of the target nucleic acid and at least one C (cytosine) or G (guanine)
That is, the base sequence of the probe is designed so that bases exist.

Preferably, when the nucleic acid probe hybridizes to the target nucleic acid, a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair in the donor dye-labeled portion. Second, the base sequence of the probe is designed. More preferably, the donor dye labeling site is a G (guanine) or C (cytosine) base, or a phosphate group of a nucleotide having the base, or an OH group of ribose.

The labeling site of the donor dye and the acceptor dye may be, when the donor dye labels the base, phosphate or ribose at the 5 ′ end of the nucleic acid probe, when the acceptor dye is in the nucleic acid probe chain or It is the 3 'terminal (including the 3' terminal base). In addition, the donor dye is a base, at the 3 'end of the nucleic acid probe,
When the phosphate moiety or the ribose moiety is labeled, the acceptor dye is in the strand of the nucleic acid probe or at the 5 ′ end (including the 3 ′ terminal base). In the case of labeling the terminal portion, labeling may be performed with both dyes. That is, for example, one of the two may be labeled with a phosphate moiety, and the other may be labeled with a ribose moiety or a base moiety (for example, when the base distance between the labeled moiety of the donor dye and the acceptor dye is 0 (described below). Described.)). Alternatively, both may be bonded to a single spacer using a spacer having a side chain.
In the present invention, it is a matter of course that the above conditions may be satisfied even if both the donor dye and the acceptor dye are labeled in the chain. It is preferable that the binding position of the fluorescent dye at each of the above-mentioned sites is bonded to an OH group or an amino group via a spacer.

The distance between the bases in the labeled portion of the donor dye and the acceptor dye essentially depends on the type of the dye pair of the donor dye and the acceptor dye, but generally ranges from 0 to 50.
Bases, preferably 0-40 bases, more preferably 0-3 bases
5 bases, particularly preferably 0 to 15 bases. If it exceeds 50 bases, the FRET phenomenon becomes unstable. With 15 to 50 bases, the fluorescence intensity of the acceptor dye decreases, but the fluorescence intensity of the donor dye may increase. That is, when the nucleic acid probe hybridizes to the target nucleic acid, the three-dimensional structure of the nucleic acid probe may change. Then, the change may eliminate the FRET phenomenon between the dyes of the nucleic acid probe. Depending on the quenching action (quenching action) of the acceptor dye on the donor dye and the magnitude of the action of the GC hydrogen bond on the donor dye, the fluorescence intensity of the donor dye increases or decreases compared to before the hybridization. . The quenching effect of the hydrogen bond of GC is decreased when the quenching effect is larger, but increased when the quenching effect is smaller. When the number of bases is less than 15, the fluorescence intensity of both the donor dye and the acceptor dye decreases after hybridization. The amount of reduction does not depend on the three-dimensional structure of the nucleic acid probe after hybridization, but only on the concentration of the target nucleic acid in the measurement reaction system. When the fluorescence intensity of both the donor dye and the acceptor dye is reduced after hybridization, the number of bases is particularly preferably 0 to 10 bases.

Therefore, a particularly preferred form of the probe of the present invention has a distance between the bases of the labeled part of the donor dye and the acceptor dye, and the donor dye is BODIPY FL or BODIPY 493.
/ 503, 5-FAM, Tetramethylrhodamine, or 6-TAMRA
And the acceptor dye is BODIPY 581/591, X-rhodamine and the 5 ′ terminal base is G or C and the 5 ′ terminal is labeled with a donor dye, or the 3 ′ terminal base of the nucleic acid probe Is G or C, and the 3 ′ end is labeled with a donor dye.

The structure of the novel nucleic acid probe for nucleic acid measurement of the present invention is as described above. With such a structure, the energy of the excited donor dye is transferred to the acceptor dye when it is not hybridized to the target nucleic acid, so that the acceptor dye emits light and has fluorescence intensity. . Thus, since the emission of the donor dye is also suppressed, the fluorescence intensity is kept at a low level. However, when the nucleic acid probe hybridizes to the target nucleic acid, the energy of the donor dye is transferred to the GC hydrogen bond generated by the probe-nucleic acid hybrid complex. In some cases, the three-dimensional structure of the nucleic acid probe changes to eliminate the FRET phenomenon. Thus, the emission of the acceptor dye is reduced.

The oligonucleotide of the nucleic acid probe of the present invention can be produced by a general method for producing an oligonucleotide. For example, chemical synthesis methods, plasmid vectors,
It can be produced by a microbial method using a phage vector or the like (Tetrahedron letters, 22, 1859-1862, 1981).
Year; Nucleic acids Research, Vol. 14, pp. 6227-6245, 1
986). It is preferable to use a commercially available nucleic acid synthesizer (for example, ABI394 (manufactured by Perkin Elmer, USA)).

For labeling a fluorescent dye on an oligonucleotide, a desired one of conventionally known labeling methods can be used (Nature Biotechnology, vol. 14, 303-3).
08, 1996; Applied and Environmental Microbiol
ogy, 63, 1143-1147, 1997; Nucleic acids
Research, 24, 4532-4535, 1996). For example, when a fluorescent dye molecule is bound to the 5 'end, first, for example,-(CH ₂ ) _n -SH is introduced as a spacer into the phosphate group at the 5' end according to a conventional method. These inductants are commercially available and may be purchased commercially (Medland Certified Reagent Company, Inc.
and Certified Reagent Company)). In this case, n is 3 to 8, preferably 6. A labeled oligonucleotide can be synthesized by binding a fluorescent dye having SH group reactivity or a derivative thereof to the spacer.
The thus synthesized oligonucleotide labeled with a fluorescent dye can be purified by reverse phase chromatography or the like to obtain the nucleic acid probe of the present invention.

[0038] The 3 'terminal base of the oligonucleotide can also be labeled. In this case, as a spacer, for example,-(CH ₂ ) _n -NH ₂ is introduced into the OH group at the 3′-position C of ribose or deoxyribose. Since these transfectants are also commercially available in the same manner as described above, commercial products may be purchased (Midland Certified Reagent Company). Further, a phosphate group is introduced, and, for example,-(CH ₂ ) _n -SH is introduced as a spacer to the OH group of the phosphate group. In these cases, n is 3-8, preferably 4-7. A labeled oligonucleotide can be synthesized by binding a fluorescent dye or a derivative thereof reactive to an amino group or SH group to this spacer.

Further, the base in the oligonucleotide chain can be labeled. The amino or OH group of the base may be labeled with the dye of the present invention in the same manner as in the method for the 5 ′ or 3 ′ end (ANALYTICAL BIOCHEMISTRY 225, pp. 32-38 (1998
Year)). When introducing an amino group, kit reagents (for example, Uni-link aminomodifier (manufactured by CLONTECH, USA),
Fluo Reporter Kit F-6082,
It is convenient to use F-6083, F-6084, and F-10220 (all manufactured by Molecular Probes, USA). Then, a fluorescent dye molecule can be bound to the oligoribonucleotide according to a conventional method. The oligonucleotide synthesized in this way is
The nucleic acid probe of the present invention can be purified by reversed phase chromatography or the like.

As described above, the nucleic acid probe of the present invention can be prepared. When the prepared nucleic acid probe for nucleic acid measurement of the present invention hybridizes to a target nucleic acid, the fluorescence intensity of the emitted light of the probe-nucleic acid hybrid complex is significantly reduced as compared to before the complex is formed. Then, a plurality of acceptor dyes can be combined with one kind of donor dye, so that the number of nucleic acid probes corresponding to the combination can be obtained. Moreover, it has the above properties. In an actual nucleic acid measurement, a decrease in the fluorescence intensity of the acceptor dye is measured, so that multiple types of nucleic acid probes can be used simultaneously with one excitation wavelength. This means that, when multiple types of target nucleic acids are present in the same measurement system, if such nucleic acid probes are simultaneously added, multiple types of nucleic acids can be measured simultaneously with one excitation wavelength. That is, many types of nucleic acids can be measured simultaneously with a simple device.

The second invention of the present invention is a method for measuring a target nucleic acid by using the nucleic acid probe prepared as described above. That is, in a measurement system in which at least one or more target nucleic acids are present, the nucleic acid measurement nucleic acid probe according to at least any one of the above 1 to 10 of the present invention, for measuring a nucleic acid that hybridizes to the target nucleic acid. A nucleic acid probe, and a nucleic acid measuring probe having at least the same number of species as the number of species of the target nucleic acid and a nucleic acid measuring probe having a different fluorescent color, and allowing the nucleic acid measuring probe to hybridize with the target nucleic acid; A method for measuring nucleic acid, characterized in that the amount of decrease in fluorescence intensity at a measurement wavelength before and after hybridization is measured at least as many wavelengths as the number of species. In addition, the measurement kit, a data analysis method for the measurement, the measurement device,
And a computer-readable recording medium in which a procedure for causing a computer to execute a data analysis process is recorded as a program.

In the present invention, the “measurement system in which at least one or more target nucleic acids are present” means that one or more target nucleic acids are present in the measurement system. Further, the “nucleic acid probe for nucleic acid measurement according to any one of the above items 1 to 10 of the present invention” has the following meaning. In the present invention, when a single type of target nucleic acid is present in the measurement system, the type of the nucleic acid measurement nucleic acid probe to be present in the measurement system may be one or more. The expression “a plurality of nucleic acids may be used” means that a plurality of nucleic acid probes for nucleic acid measurement that hybridize to a single target nucleic acid at a plurality of sites having different base sequences may be used.

In the present invention, when a plurality of types of target nucleic acids are present, at least a plurality of types of nucleic acid probes for nucleic acid measurement are present. And the number of the types is at least the same as the number of the target nucleic acid types. And `` at least the same number '' means that a kind of target nucleic acid is provided with a base sequence site where a plurality of kinds of nucleic acid measuring probes hybridize, and a plurality of kinds of nucleic acid measuring probes are hybridized with one kind of target nucleic acid. This means that it may be present in the measurement system. Further, the term “plurality of nucleic acid measurement probes” when the single and multiple types of target nucleic acids are present in the measurement system has the following meaning. That is, (1) a plurality of different types of probes among the nucleic acid probes for nucleic acid measurement according to any one of the above items 1 to 10 of the present invention. For example, a probe having the same form or structure but different dyes for labeling each other can be exemplified. (2) A combination of probes for nucleic acid measurement having different forms or structures of probes, but a plurality of types of nucleic acid measurement probes having different labeling dyes. In other words, it means that a nucleic acid probe extending over each of the above items 1 to 10 may be used. For example, a combination of one kind of probe among the kinds of probes described in the above 5, and one kind of probe among the kinds of the probes described in the above 10 and 10, It refers to a plurality of types of nucleic acid measurement probes having different labeling dyes (the present invention is not limited by these examples). The same applies to the following description.

According to a third aspect of the present invention, there is provided a method for analyzing or measuring polymorphism and / or mutation of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present. The nucleic acid probe according to at least one of the above, wherein at least the same number of species as the number of species of the target nucleic acid, and a nucleic acid measurement probe having a different fluorescent color is present in the measurement system,
A target nucleic acid is hybridized with a probe for nucleic acid measurement, and the amount of decrease in fluorescence intensity at the measurement wavelength before and after hybridization is measured at at least the same number of wavelengths as the number of species of the probe for nucleic acid measurement. A method for analyzing or measuring a polymorphism or / and mutation of a target nucleic acid, a measurement kit thereof, a data analysis method for the measurement, a measurement device thereof, and a procedure for causing a computer to execute a data analysis process as a program The recorded computer-readable recording medium.

That is, the nucleic acid probe of the present invention can be used not only for nucleic acid measurement but also for polymorphism of a target nucleic acid.
Or / and / or a method for analyzing or measuring mutation. In particular, a DNA chip described below (protein nucleic acid enzyme, vol. 43, 2004-201)
1 page, 1998) to provide a convenient method. That is, in the hybridization with the nucleic acid probe of the present invention, the fluorescence intensity changes depending on whether or not a GC pair is formed. Then, by hybridizing the nucleic acid probe of the present invention to the target nucleic acid and measuring the emission intensity, the polymorphism (polymorp) of the target nucleic acid is measured.
hism) or / and mutations can be analyzed or measured. The specific method is described in Examples. In this case, the target nucleic acid may be an amplified product amplified by one of various nucleic acid amplification methods, or may be an extracted product. The type of the target nucleic acid is not limited. However, a guanine base or cytosine base may be present in the chain or at the terminal. If no guanine base or cytosine base is present in the chain or at the end, the fluorescence intensity does not decrease. Therefore, according to the method of the present invention, G → A, G ←
A, C → T, C ← T, G → C, G ← C mutation or substitution, ie, single nucleotide polymorphism (single nucleotide polymo
rphism (SNP)) and other polymorphisms can be analyzed or measured. At present, polymorphism analysis is currently performed by determining the base sequence of a target nucleic acid using the Maxam-Gilbert method and the dideoxy method.

By including the nucleic acid probe of the present invention in a measurement kit for analyzing or measuring polymorphism and mutation of a target nucleic acid,
It can be suitably used as a measurement kit for analyzing or measuring polymorphism and / or mutation of a target nucleic acid.

Polymorphism of the target nucleic acid of the present invention
And / or when analyzing data obtained by a method of analyzing or measuring mutation, the fluorescence intensity value of the reaction system when the target nucleic acid hybridizes with the nucleic acid probe of the present invention, By providing a process of correcting the fluorescence intensity value of the reaction system when the soybean is not soyed, the processed data becomes highly reliable. Therefore, the third invention relates to a polymorphism (polymorp
data analysis methods for analyzing or measuring hism) or / and mutations.

Further, the third invention is directed to a polymorphism (poly
In a measuring device for analyzing or measuring morphism or / and mutation, the fluorescence intensity value of the reaction system when the target nucleic acid is hybridized with the nucleic acid probe of the present invention, And a means for analyzing or measuring polymorphism and / or mutation of a target nucleic acid, comprising means for performing a correction process based on the fluorescence intensity value of the reaction system.

Further, the third invention is directed to a polymorphism (poly
When analyzing data obtained by a method for analyzing or measuring morphism or / and mutation, the fluorescence intensity value of the reaction system when the target nucleic acid is hybridized with the nucleic acid probe of the present invention is determined as described above. Is a computer-readable recording medium in which a procedure for causing a computer to execute a process of correcting with a fluorescence intensity value of a reaction system when hybridization has not occurred is recorded as a program.

Further, the third invention is that a plurality of the nucleic acid probes for nucleic acid measurement of the present invention are bound to the surface of a solid support, and the target nucleic acid is hybridized thereto to measure the target nucleic acid. A device (chip) for measuring the concentration of a plurality of nucleic acids, each of which is characterized by a nucleic acid probe arranged in an array on the surface of a solid support and bonded to each other. In addition, for each nucleic acid probe bound to the surface of the solid support, at least one temperature sensor and a heater are installed on the opposite surface, and the temperature can be adjusted so that the nucleic acid probe binding region has an optimal temperature condition. It is.

That is, the nucleic acid probe of the present invention can be immobilized on the surface of a solid (support layer), for example, the surface of a slide glass. This format is now called a DNA chip. Monitoring of gene expression,
Determination of base sequence, mutation analysis (mutati
on analysis) single nucleotide polym
orphism (SNP))
is). Of course, it can also be used as a device (chip) for nucleic acid measurement.

For binding the nucleic acid probe of the present invention to, for example, the surface of a slide glass, a slide glass coated with a polycation such as polylysine, polyethyleneimine or polyalkylamine, a slide glass into which an aldehyde group is introduced, or an amino group can be used. First, the introduced slide glass is prepared. And, i) react the phosphate group of the probe with the slide glass coated with the polycation, ii)
An aldehyde group-introduced glass slide is reacted with an amino group-introduced probe. Iii) An amino group-introduced glass slide is subjected to PDC (pyridinium dichroma).
ate) introduction, and reaction with a probe introduced with an amino group or an aldehyde group, and the like (Fodor, PA,
et al., Science, 251, 767-773 (1991); Schena, M., et a
l., Proc. Natl. Acad. Sci., USA, 93, 10614-10619 (199
6); McGal, G., et al., Proc. Natl. Acad. Sci., USA, 93, 1
3555-13560 (1996); Blanchard, AP, et al., Biosens. Bioe
lectron., 11,687-690 (1996)).

The device in which the nucleic acid probes for nucleic acid measurement of the present invention are arrayed and bound on a solid surface in an array makes nucleic acid measurement more convenient. In this case, by making a device in which many nucleic acid probes of the present invention having different base sequences are individually bound on the same solid surface, multiple types of target nucleic acids can be measured at the same time. In this device, for each probe, at least one temperature sensor and a heater are provided on the surface opposite to the surface to which the solid nucleic acid probe of the present invention is bonded, and the region of the solid to which the probe is bonded is set to an optimum temperature condition. It is particularly preferred that it is designed so that it can be temperature-controlled so that

The basic operation of the measuring method using the device of the present invention is only to place a solution containing a target nucleic acid on a solid surface to which a nucleic acid probe is bound and to hybridize. As a result, the amount of fluorescence changes in accordance with the amount of the target nucleic acid, and the target nucleic acid can be measured from the amount of change in the fluorescence. In addition, by binding a large number of nucleic acid probes of the present invention having different base sequences on one solid surface, the concentration of many target nucleic acids can be measured at once. Therefore, it is a novel DNA chip because it can be used for the measurement of a target nucleic acid in exactly the same application as the DNA chip. Under optimal reaction conditions, nucleic acids other than the target nucleic acid do not change the amount of fluorescence emitted, so that there is no need to perform an operation to wash unreacted nucleic acids.
Further, by controlling the temperature of each nucleic acid probe of the present invention with a micro heater, it is possible to control the reaction conditions to the optimum for each probe, so that accurate measurement of the concentration becomes possible. Further, the dissociation curve between the nucleic acid probe of the present invention and the target nucleic acid can be analyzed by continuously changing the temperature with a micro heater and measuring the amount of fluorescence during that time. From the difference in the dissociation curves, the properties of the hybridized nucleic acid can be determined, and the SNP can be detected.

A conventional device for measuring the concentration of a target nucleic acid is as follows.
A nucleic acid probe not modified with a fluorescent dye is bound (fixed) to a solid surface, and a target nucleic acid labeled with a fluorescent dye is hybridized with the solid surface. The fluorescence intensity of the fluorescent dye was measured. To label a target nucleic acid with a fluorescent dye, for example, when targeting a specific mRNA, the following steps are taken: (1) m extracted from cells
Extract all RNA. (2) Then, cDNA is synthesized using reverse transcriptase while incorporating a nucleoside modified with a fluorescent dye. In the present invention, such an operation is not necessary.

The device has a large number of spots of various types of probes, but the optimal hybridization conditions for nucleic acids hybridizing to each probe, such as the temperature, are different. Therefore, the hybridization reaction, the probe,
It is necessary to perform the washing operation under optimal conditions. However, since it is physically impossible, hybridization is carried out at the same temperature for all probes, and washing is carried out at the same temperature with the same washing liquid. Therefore, the nucleic acid expected to be hybridized has a drawback that it does not hybridize, and even if hybridized, the nucleic acid is not strong enough to be easily washed away. Due to such reasons, the quantification of nucleic acids was low. The present invention does not have such a disadvantage because this washing operation is not required. Further, by providing a micro heater at the bottom of the spot and controlling the hybridization temperature, a hybridization reaction can be performed at an optimum temperature for each probe of the present invention. Therefore, in the present invention, the quantitativeness is significantly improved.

In the present invention, by using the nucleic acid probe or device for nucleic acid measurement of the present invention described above, a target nucleic acid can be easily and specifically identified in a measurement system in which at least one or more target nucleic acids are present. Can be measured. The measurement method is described below. In the measurement method of the present invention, first, a nucleic acid probe corresponding to the target nucleic acid for nucleic acid measurement of the present invention is added to the measurement system at least as many as the number of species of the target nucleic acid, and hybridized to the target nucleic acid. . The method can be carried out by a commonly known method (Analytical Biochemistry, 183, 231).
244, 1989; NatureBiotechnology, 14, 303
308, 1996; Applied and Environmental Microb
iology, 63, 1143-1147, 1997). For example, hybridization conditions are such that the salt concentration is 0 to 2 molar, preferably 0.1 to 1.0 molar, and the pH is 6 to
8, preferably 6.5 to 7.5.

The reaction temperature is preferably in the range of Tm ± 10 ° C. of the nucleic acid hybrid complex obtained by hybridizing the nucleic acid probe for nucleic acid measurement of the present invention to a specific site of the target nucleic acid. By doing so, non-specific hybridization can be prevented. When the temperature is lower than Tm-10 ° C, non-specific hybridization occurs. When the temperature exceeds Tm + 10 ° C, no hybridization occurs. The Tm value can be determined in the same manner as in the experiment necessary for designing the nucleic acid probe for measuring a nucleic acid of the present invention. That is, an oligonucleotide (having a base sequence complementary to the nucleic acid probe) that hybridizes with the nucleic acid probe for nucleic acid measurement of the present invention is chemically synthesized using the nucleic acid synthesizer or the like, and the nucleic acid hybrid complex with the nucleic acid probe is synthesized. The body Tm value is measured in the usual way. When a plurality of target nucleic acids are present in the measurement system, an average value of a plurality of nucleic acid hybrid complexes may be employed, or a target nucleic acid which is most regarded as important may be employed.

The reaction time is 1 second to 180 minutes, preferably 5 seconds to 90 minutes. When the time is shorter than 1 second, the number of unreacted nucleic acid probes of the present invention in hybridization increases. Further, it is meaningless if the reaction time is too long. The reaction time is greatly affected by the nucleic acid species, that is, the length of the nucleic acid or the base sequence. As described above, the nucleic acid probe for nucleic acid measurement of the present invention is hybridized to the target nucleic acid. Before and after hybridization, the fluorescence intensity of the hybridization reaction system is measured with a fluorometer at at least the same number of wavelengths as the number of species of the added nucleic acid measurement probe, and the amount of decrease at each wavelength is measured. Is calculated. Since the magnitude of the decrease is proportional to the concentration of the target nucleic acid, the concentration of the target nucleic acid can be determined.

The concentration of the target nucleic acid in the reaction solution: 0.1 to 1
Preferably it is 0.0 nM. The concentration of the nucleic acid probe of the present invention in the reaction solution is preferably 1.0 to 25.0 nM. When preparing a calibration curve, it is desirable to use the nucleic acid probe of the present invention at a ratio of 1.0 to 2.5 with respect to the target nucleic acid.

In practice, when measuring an unknown concentration of a target nucleic acid in a sample, a calibration curve is first prepared under the above conditions. When there are a plurality of target nucleic acids, a calibration curve is created for each measurement wavelength of each nucleic acid probe of the present invention corresponding to the target nucleic acid. Then, the nucleic acid probe of the present invention corresponding to a plurality of concentrations is added to the sample, and the decrease in the fluorescence intensity value is measured. Then, the probe concentration corresponding to the largest one of the measured decrease values of the fluorescence intensity is set as a preferable probe concentration. The quantitative value of the target nucleic acid is determined from the calibration curve based on the decrease in the fluorescence intensity measured with the probe at the preferred concentration.

The fourth invention of the present invention is directed to a method for measuring various nucleic acids, for example, a FISH method, a PCR method, and an LCR method.
Method, SD method, competitive hybridization method,
The invention is applied to the TAS method, and the like.

An example is described below. a) When applied to the FISH method. That is, the present invention relates to a microorganism system in which various types of microorganisms are mixed or one or more types of microorganisms are mixed with cells derived from animals or plants and cannot be isolated from each other. The present invention can be suitably applied to nucleic acid measurement of intracellular or homogenate of cells in complex microbial systems and symbiotic microbial systems). The microorganism here is a microorganism generally referred to, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms, mycoplasmas, viruses, rickettsies, and the like can be mentioned. The target nucleic acid in this system is, for example, a nucleic acid having a base sequence having specificity for cells of a bacterial strain to be examined for its activity in these microorganism systems. For example, 5SrR of a specific strain
Specific sequence of NA, 16S rRNA or 23S rRNA or their gene DNA.

The nucleic acid probe of the present invention is added to a complex microbial system or a symbiotic microbial system to obtain 5S rRNA,
By measuring the amount of 6S rRNA or 23S rRNA or their gene DNA, the abundance of a specific strain in the system can be measured. In addition, a method of adding the nucleic acid probe to a homogenate of a complex microbial system or a symbiotic microbial system, measuring the amount of decrease in luminescence of a fluorescent dye before and after hybridization, and measuring the abundance of a specific strain is also disclosed in the present invention. Within the target range.

The above measuring method is, for example, as follows. That is, before adding the nucleic acid probe of the present invention, the temperature, salt concentration and pH of the complex microorganism system or symbiotic microorganism system are adjusted to the above-mentioned conditions. The specific strain in the complex microbial system or the symbiotic microbial system is adjusted to a cell number of 10 ⁷ to 10 ¹² cells / ml, preferably 10 ⁹ to 10 ¹⁰ cells / ml. It can be carried out by dilution or concentration by centrifugation or the like. Number of cells is 10 ⁷ cells / ml
When it is less than the above, the fluorescence intensity is weak and the measurement error becomes large. When it exceeds 10 ¹² cells / ml, the fluorescence intensity of the complex microorganism system or the symbiotic microorganism system is too strong, so that the abundance of the specific microorganism cannot be quantitatively measured.

The concentration of the nucleic acid probe of the present invention to be added is
It depends on the cell number of a particular strain in a complex or commensal microbial system. 0.1 for a cell count of 1 × 10 ⁸ / ml.
The concentration is 1 to 10.0 nM, preferably 0.5 to 5 nM, more preferably 1.0 nM. 0.1 nM
If the value is less than 1, the data does not accurately reflect the abundance of the specific microorganism. However, the optimal amount of the nucleic acid probe of the present invention cannot be determined unconditionally because it depends on the amount of the target nucleic acid in the cell.

Next, in the present invention, the nucleic acid probe and 5S rRNA, 16S rRNA or 23S rRNA of a specific strain are used.
The reaction temperature when hybridizing to rRNA or its gene DNA is the same as the above-mentioned conditions. The reaction time is also the same as the above conditions. Under the above conditions, the nucleic acid probe of the present invention is
The DNA is hybridized to NA, 16S rRNA or 23S rRNA or its gene DNA, and the amount of decrease in the emission of the fluorescent dye of the complex microorganism or the symbiotic microorganism before and after hybridization is measured.

The amount of decrease in the emission of the fluorescent dye measured as described above is proportional to the amount of the specific strain present in the complex microorganism system or the symbiotic microorganism system. It is 5S rRNA,
This is because the amount of 16S rRNA or 23S rRNA or their gene DNA is proportional to the amount of the specific strain.

In the present invention, the components other than the microorganism in the complex microbial system or the symbiotic microbial system are the nucleic acid probe of the present invention and 5SrRNA or 16SrRN of a specific strain.
A or 23S rRNA or their gene DNA
Unless it inhibits hybridization with
The nucleic acid probe of the present invention is not particularly limited as long as it does not inhibit the emission of the fluorescent dye. For example, KH ₂ PO ₄ , K ₂ HPO ₄ , Na
Phosphates such as H ₂ PO ₄ and Na ₂ HPO ₄ ; inorganic nitrogens such as ammonium sulfate, ammonium nitrate and urea; various salts of metal ions such as magnesium, sodium, potassium and calcium ions; manganese, zinc, iron and cobalt ions Sulfates of trace metal ions such as
Various salts such as hydrochloride and carbonate, and vitamins may be appropriately contained. If the above-mentioned inhibition is observed, cells may be separated from a cell line in which a plurality of microorganisms are mixed by an operation such as centrifugation and suspended again in a buffer solution or the like.

As the above buffer, various buffers such as a phosphate buffer, a carbonate buffer, a Tris / HCl buffer, a Tris / glycine buffer, a citrate buffer, and a Goodt buffer can also be used. . The concentration of the buffer is a concentration that does not inhibit the hybridization, the FRET phenomenon, and the emission of the fluorescent dye. The concentration depends on the type of buffer. The pH of the buffer is 4-12, preferably 5-9.

B) When applied to the PCR method Any method can be applied as long as it is a PCR method. The case where the method is applied to a real-time quantitative PCR method is described below. That is, in a real-time quantitative PCR method, the nucleic acid probe for nucleic acid measurement of the present invention is used
The CR is performed, and the decrease in the fluorescence intensity of the reaction system before and after the reaction is measured in real time. The PCR of the present invention means PCR by various methods. For example, RT-
It also includes PCR, RNA-primed PCR, Stretch PCR, inverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, PCR using PNA, and the like. Further, the term “quantitative” means quantitative measurement of the degree of detection in addition to the original quantitative measurement, as described above.

As described above, the target nucleic acid refers to a nucleic acid whose amount is to be measured. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids may be mixed. For example, a specific nucleic acid to be amplified in a complex microbial system (mixed system of RNA or gene DNA of multiple microorganisms) or a symbiotic microbial system (mixed system of RNA or gene DNA of multiple animals and plants and / or multiple microorganisms). If the target nucleic acid needs to be purified, it can be performed by a conventionally known method. For example, it can be performed using a commercially available purification kit or the like.

Conventionally known quantitative PCR methods include dATP,
Using dGTP, dCTP, dTTP or dUTP, a target nucleic acid (DNA or RNA), Taq polymerase, a primer, and a nucleic acid probe or an intercalator labeled with a fluorescent dye, in the presence of Mg ions, the temperature is repeatedly reduced and increased. It amplifies a target nucleic acid and monitors the amount of increase in luminescence of a fluorescent dye in the amplification process in real time (Experimental Medicine, Vol. 15, 7).
No. 46-51, 1997, Youtosha).

In the quantitative PCR method of the present invention, when amplifying at least one or more target nucleic acids, the target nucleic acids are amplified by using at least the same number of nucleic acid probes of the present invention as the number of target nucleic acid species. And in the amplification process,
The amount of decrease in the fluorescence intensity of the reaction system is measured at least as many wavelengths as the number of species of the nucleic acid probe. In the quantitative PCR of the present invention, any of the nucleic acid probes for nucleic acid measurement of the present invention can be suitably used, but the number of bases is 5 to 50, preferably 10 to 10.
-25, particularly preferably 15-20, as long as they hybridize with the amplification product of the target nucleic acid during the PCR cycle. Also, forward (fo
rward) type or reverse type.

Preferred nucleic acid probes include:
The following can be mentioned. (1) When a nucleic acid probe hybridizes to a target nucleic acid, a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair in the donor dye-labeled portion.
That is, the nucleotide sequence of the probe is designed.
More preferably, (2) In the above (1), the donor dye labeling site is a G (guanine) or C (cytosine) base, a phosphate group of a nucleotide having the base, or an O group of ribose.
H group. The acceptor dye is at the 3 'end or in the chain without the 3' terminal base. (3) In the above (2), the acceptor dye is a nucleic acid probe that is labeled closer to the donor dye labeling site in the chain.

Among the nucleic acid probes of the present invention, (1) when the donor dye labels the 5 ′ terminal base or 5 ′ terminal base of the nucleic acid probe, the acceptor dye is in the nucleic acid probe chain or at the 3 ′ terminal. The nucleic acid probe labeling a part of bases is one that can be used as a primer.

When used as this primer, if the 3 ′ or 5 ′ end cannot be designed to be G or C from the base sequence of the target nucleic acid, the 5 ′ of the oligonucleotide designed as a primer based on the base sequence of the target nucleic acid must be used. Even if 5'-guanylic acid or guanosine or 5'-cytidylic acid or cytidine is added to the terminal, the object of the present invention can be suitably achieved. Even if 5'-guanylic acid or 5'-cytidylic acid is added to the 3 'end, the object of the present invention can be suitably achieved. Therefore, in the present invention,
A nucleic acid probe designed so that the base at the 3 ′ or 5 ′ end is G or C is a probe designed from the base sequence of a target nucleic acid, as well as a 5 ′ at the 5 ′ end of the probe.
A probe comprising guanylic acid or guanosine or 5'-cytidylic acid or cytidine, and a probe comprising 5'-guanylic acid or 5'-cytidylic acid at the 5 'end of the probe. Define.

Further, among the nucleic acid probes of the present invention,
(2) When the donor dye labels the 3 ′ terminal base (including the 3 ′ terminal base) or the 3 ′ terminal ribose or deoxyribose of the nucleic acid probe, the acceptor dye may be in the nucleic acid probe chain or 5 ′. 'The nucleic acid probe labeling the base at the terminal is designed so as not to be used as a primer. PCR is performed using one nucleic acid probe of the present invention instead of two probes (labeled with a fluorescent dye) used in the real-time quantitative PCR method using the FRET phenomenon. The probe is added to the PCR reaction system, and PCR is performed. During the nucleic acid extension reaction, the probe hybridized to the target nucleic acid or the amplified target nucleic acid is decomposed by the polymerase and decomposed and removed from the nucleic acid hybrid complex. At this time, the fluorescence intensity value of the reaction system or the reaction system in which the nucleic acid denaturation reaction is completed is measured. In addition, the fluorescence of a reaction system in which the target nucleic acid or the amplified target nucleic acid is hybridized with the probe (reaction system during an annealing reaction or a nucleic acid extension reaction until the probe is removed from the nucleic acid hybrid complex by a polymerase). Measure the intensity value. Then, the amplified nucleic acid is measured by calculating the decrease rate of the fluorescence intensity value from the former. Fluorescence when the probe is completely dissociated from the target nucleic acid or amplified target nucleic acid by a nucleic acid denaturation reaction or when degraded and removed from the nucleic acid hybrid complex of the probe and the target nucleic acid or amplified target nucleic acid by polymerase during nucleic acid extension The intensity values are large. However, a nucleic acid hybrid complex between the probe and the target nucleic acid or the amplified target nucleic acid by a polymerase during an annealing reaction or a nucleic acid extension reaction in which the annealing reaction is completed or the probe is sufficiently hybridized to the target nucleic acid or the amplified target nucleic acid The fluorescence intensity value of the reaction system until it is decomposed and removed from is lower than the former. The decrease in the fluorescence intensity value is proportional to the amount of the amplified nucleic acid.

In this case, when the probe is hybridized with the target nucleic acid, the T
m is in the range of ± 15 ° C., preferably ± 5 ° C. of the Tm value of the nucleic acid hybrid complex of the primer,
It is desirable that the nucleotide sequence of the probe (2) be designed. The Tm value of the probe is the Tm value of the primer −5 ° C.
In particular, when the temperature is lower than −15 ° C., since the probe does not hybridize, the emission of the fluorescent dye does not decrease. Conversely, if the Tm value of the primer exceeds + 5 ° C., especially + 15 ° C., the probe hybridizes to nucleic acids other than the target nucleic acid, and the specificity of the probe is lost.

Probes other than the above (2), particularly the probe (1), are added to the PCR reaction system as primers. A PCR method using a primer labeled with a fluorescent dye has not yet been known except for the present invention. PCR
As the reaction proceeds, the amplified nucleic acid is secondarily labeled with a fluorescent dye useful for practicing the present invention. Therefore, the fluorescence intensity value of the reaction system in which the nucleic acid denaturation reaction is completed is large, but in the reaction system in which the annealing reaction is completed or the nucleic acid extension reaction is performed, the fluorescence intensity of the reaction system is lower than the former fluorescence intensity. I do.

The PCR reaction can be performed under the same reaction conditions as in the ordinary PCR method. Thus, the target nucleic acid can be amplified in a reaction system in which the Mg ion concentration is low (1-2 mM). Of course, the present invention can also be carried out in a reaction system in the presence of a high concentration (2 to 4 mM) of Mg ions used in conventionally known quantitative PCR.

In the PCR method of the present invention, the PCR of the present invention is carried out, and the nucleic acid melting curve of the amplification product is analyzed at least at the same number of wavelengths as the number of species of the probe of the present invention used. To determine the Tm value of the target nucleic acid. This method is a novel method for analyzing a melting curve of a nucleic acid. In the present method, the nucleic acid probe used in the PCR method of the present invention or the nucleic acid probe of the present invention used as a primer can be suitably used. In this case, by making the base sequence of the nucleic acid probe of the present invention a sequence complementary to a region containing SNP (snip; single nucleotide substitution polymorphism),
After completion of the CR, the dissociation curve of the nucleic acid from the nucleic acid probe of the present invention is analyzed, whereby S
NP can be detected. If a nucleotide sequence complementary to a sequence containing SNP is used as the sequence of the nucleic acid probe of the present invention,
The Tm value obtained from the dissociation curve between the probe sequence and the sequence containing SNP is higher than the Tm value obtained from the dissociation curve between the probe sequence and the sequence containing no SNP.

The fifth invention of the present invention is a method for analyzing data obtained by the above-mentioned real-time quantitative PCR method. Real-time quantitative PCR methods are currently
A reaction device for performing PCR, a device for detecting luminescence of a fluorescent dye, a user interface, that is, a computer-readable recording medium (separate name: Sequence Detec) in which each procedure of the data analysis method is programmed and recorded.
Measurement Software System) and a device consisting of a computer that controls them and analyzes data, and is measured in real time. Thus, the measurement according to the invention is also performed with such a device.

Hereinafter, first, real-time quantitative PCR
The analysis device will be described first. The device used in the present invention may be any device as long as it can monitor PCR in real time. For example, ABI PRISM
^TM 7700 Sequence Detection System
ystem SDS 7700) (Perkin Elmer Applied Biosytems, Inc.
USA)) and LightCycler ^™ system (Roche Diagnostics, Germany).

The above-mentioned PCR reaction apparatus is an apparatus for repeatedly performing a thermal denaturation reaction, an annealing reaction, and an elongation reaction of a target nucleic acid (for example, at a temperature of 95 ° C., 60 ° C., 72 ° C.).
Can be repeated. ). The detection system is composed of an argon laser for fluorescence excitation, a spectrograph, and a CCD camera. Further, each procedure of the data analysis method is programmed, and a computer-readable recording medium recording the program is installed and used in a computer, controls the above system via the computer, and is output from the detection system. It records a program for analyzing and processing data.

The data analysis program recorded on the computer-readable recording medium is a process for measuring the fluorescence intensity for each cycle, and measures the measured fluorescence intensity as a function of the cycle, that is, PCR amplification.
The process of displaying a plot as a plot on a computer display, the number of PCR cycles (thre
(shold cycle number: Ct), a step of preparing a calibration curve for obtaining the copy number of the sample nucleic acid from the Ct value, and a step of printing data and plot values of each step. If the PCR is progressing exponentially,
A linear relationship holds between the Log value of the copy number of the target nucleic acid at the start and Ct. Therefore, by preparing a calibration curve using a known number of copies of the target nucleic acid and detecting the Ct of the sample containing the unknown number of copies of the target nucleic acid, the copy number of the target nucleic acid at the start of PCR can be calculated.

C) A method for analyzing data obtained by the PCR method, an analyzing apparatus, and a computer-readable recording medium on which an analysis procedure for causing a computer to execute the analysis is recorded as a program. The present invention is a method for analyzing data obtained by the real-time quantitative PCR method as described above. Each feature is described below. The first feature is that, in a method for analyzing data obtained by a real-time quantitative PCR method, when the amplified nucleic acid in each cycle binds to a fluorescent dye, or the amplified nucleic acid hybridizes with the nucleic acid probe of the present invention. The fluorescence intensity value of the reaction system at the time, the fluorescent dye and nucleic acid bound in each cycle, namely, a fluorescent dye-nucleic acid complex, or the nucleic acid probe of the present invention and the target nucleic acid hybridized, that is, nucleic acid This is an arithmetic processing step of correcting with the fluorescence intensity value of the reaction system when the hybrid complex is dissociated, that is, a correction arithmetic processing step.

The “reaction system when the amplified target nucleic acid binds to a fluorescent dye or when the amplified target nucleic acid hybridizes with the nucleic acid probe of the present invention” is specifically exemplified by a reaction system in each cycle of PCR. 40-85
And a reaction system at the time of nucleic acid extension reaction or annealing having a reaction temperature of 50 ° C, preferably 50 to 80 ° C. And, it means a reaction system in which the reaction is completed. The actual temperature depends on the length of the amplified nucleic acid. Further, "the reaction system when the fluorescent dye-nucleic acid complex or the nucleic acid hybrid complex is dissociated"
A reaction system for heat denaturation of nucleic acid in each cycle of PCR, specifically, a reaction temperature of 90 to 100 ° C., preferably 94 to 100 ° C.
At a temperature of 96 ° C., a system in which the reaction is completed can be exemplified.

The correction calculation process in the correction calculation process may be any process as long as it meets the object of the present invention. Specifically, the process according to the following [Formula 1] or [Formula 2] An example including a process can be exemplified. f _n = f _{hyb, n} / f _{den, n} [Formula 1] f _n = f _{den, n} / f _{hyb, n} [Formula 2] [where, f _n : calculated by [Formula 1] or [Formula 2] F _{hyb, n} : the reaction system when the amplified nucleic acid binds to the fluorescent dye or when the amplified nucleic acid hybridizes with the nucleic acid probe of the present invention in the nth cycle _{Fden, n} : the fluorescence intensity value of the reaction system when the above-described fluorescent dye-nucleic acid complex or nucleic acid hybrid complex is dissociated in the nth cycle] It includes a sub-step of displaying the corrected arithmetic processing value obtained in the processing on a computer display and / or similarly displaying and / or printing the value in the form of a graph as a function of the number of cycles.

The second feature is that the correction operation value obtained by [Equation 1] or [Equation 2] in each cycle is substituted into the following [Equation 3] or [Equation 4], and the fluorescence change ratio between the samples or This is a data analysis method that calculates the rate of change in fluorescence and compares them.

F _n = f _n / f _a [Equation 3] F _n = f _a / f _n [Equation 4] _where F _{n is} calculated by [Equation 3] or [Equation 4] in the nth cycle. Calculated fluorescence change rate or fluorescence change rate, f _n : correction calculation processing value by [Equation 1] or [Equation 2] in the nth cycle f _a : correction calculation processing value by [Equation 1] or [Equation 2] but those of the previous number of any cycle the change in f _n is observed, usually, for example, those of 10 to 40 cycles, preferably those of 15 to 30 cycles, more preferably 20 to 30 cycles Is adopted. In this process, the calculated value obtained in the above process is displayed and / or printed on a computer display, or the comparison value or the value is similarly displayed and / or graphed as a function of each cycle number. Alternatively, it includes a sub-step of printing, but the above-described sub-step may or may not be applied to the correction operation processing value by [Equation 1] or [Equation 2].

The third characteristic is that (3.1) [Expression 5] and [Expression 6] are obtained by using the fluorescence change rate or the data of the fluorescence change rate calculated by [Equation 3] or [Equation 4].
Alternatively, a process of performing an arithmetic processing by [Equation 7], log _b (F _n ), ln (F _n ) [Equation 5] log _b {(1-F _n ) × A}, ln ｛(1-F _n ) × A ｝ [Equation 6] log _b {(F _n -1) × A}, ln {(F _n -1) × A} [Equation 7]

[Wherein, A and b are arbitrary numerical values, preferably integer values, and more preferably natural numbers. When A = 100 and b = 10, {(F _n −1) × A} is expressed as a percentage (%). F _n : Fluorescence change rate or fluorescence change rate in n cycles calculated by [Equation 3] or [Equation 4]; Arithmetic process for calculating a number, (3.3)
An arithmetic processing step of calculating the relational expression between the number of cycles in the nucleic acid sample of known concentration and the number of copies of the target nucleic acid at the start of the reaction,
(3.4) A data analysis method including an arithmetic processing step of calculating the copy number of a target nucleic acid at the start of PCR in an unknown sample. Then, (3.1) → (3.2) → (3.
A process consisting of the order of 3) → (3.4) is preferred.

The steps (3.1) to (3.3) are as follows:
A sub-step of displaying and / or printing the calculated values obtained in each process on a computer display and / or displaying the values in the form of a graph as a function of the number of cycles in the same manner as above. Is also good.
Since the operation processing value obtained in the process (3.4) needs to be printed at least, the process includes a sub-step of printing. The operation processing value obtained in (3.4) may be further displayed on a display of a computer. In addition, the correction calculation processing value according to [Equation 1] or [Equation 2] and the calculation processing value according to [Equation 3] or [Equation 4] are displayed on a computer display in the form of a graph as a function of each cycle number and / or. Or even if printed,
The display and / or printing sub-steps may be added as necessary.

The above data analysis method is particularly effective when the real-time quantitative PCR method is to measure the decrease in the emission of a fluorescent dye. A specific example is the aforementioned real-time quantitative PCR method of the present invention.

The fourth feature is real-time quantitative PCR.
Is a real-time quantitative PCR measurement and / or analysis device having an operation and storage means for executing the data analysis method for the real-time quantitative PCR method of the present invention.

The fifth characteristic is real-time quantitative PCR.
The respective steps of the data analysis method for analyzing PCR using the analysis device of the present invention are programmed, and the computer executes the respective steps of the data analysis method of the present invention on a computer-readable recording medium on which the program is recorded. Is a computer-readable recording medium on which is recorded a program capable of being executed.

A sixth feature is a method for analyzing the melting curve of a nucleic acid of the present invention, that is, a method for analyzing data obtained by a method for obtaining a Tm value of a nucleic acid by performing a PCR method of the present invention. That is, for nucleic acids amplified by the PCR method of the present invention, a process of gradually increasing the temperature from a low temperature until the nucleic acid is completely denatured (for example, 50 to 95)
C.) in this process, for short time intervals (for example, 0.
Measuring the fluorescence intensity at intervals corresponding to a temperature rise of 2 to 0.5 ° C.), displaying the measurement results on a display as a function of time, ie, displaying the melting curve of the nucleic acid, and this melting curve. Is differentiated to obtain a differential value (−dF / dT,
F: fluorescence intensity, T: time), a process of displaying the value on a display as a differential value, and a process of obtaining an inflection point from the differential value. In the present invention, the fluorescence intensity increases as the temperature increases. In the present invention, during the nucleic acid extension reaction in each cycle,
Preferably, a more preferable result is obtained by adding, to the above-mentioned process, a step of performing a calculation process of dividing the fluorescence intensity value at the end of the PCR reaction using the fluorescence intensity value at the time of the thermal denaturation reaction.

An apparatus for measuring and / or analyzing real-time quantitative PCR of the present invention obtained by adding a method for analyzing a melting curve of a nucleic acid of the present invention to the data analysis method of the novel PCR method of the present invention. It is within the technical scope of the present invention.

Further, a seventh feature is that a computer-readable recording medium on which a program for causing a computer to execute each procedure of the method for analyzing a melting curve of a nucleic acid of the present invention, and The PC of the present invention
On a computer-readable recording medium on which a program that enables a computer to execute each procedure of the data analysis method of the R method is used, the procedure of the method for analyzing a melting curve of a nucleic acid of the present invention is executed by a computer. It is a computer-readable recording medium in which a program that can be executed is additionally recorded.

The data analysis method, apparatus, and recording medium of the present invention include medicine, forensic medicine, anthropology, ancient biology,
It can be used in various fields such as biology, genetic engineering, molecular biology, agriculture, and plant breeding. Also referred to as a complex microbial system, a symbiotic microbial system, a microbial system in which various types of microorganisms are mixed or at least one type of microorganism is mixed with cells derived from other animals or plants and cannot be isolated from each other. It can be used suitably. The microorganism referred to here is a microorganism generally referred to, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms, other mycoplasmas, viruses, rickets and the like can be mentioned.

Further, using one or more of the data analysis method, apparatus and recording medium of the present invention, 5SrR of a specific strain in a complex microorganism system or a symbiotic microorganism system
By quantifying the copy number of NA, 16S rRNA or 23S rRNA or their gene DNA,
The amount of the specific strain in the system can be measured. It can be 5S rRNA, 16S rRNA or 2
This is because the copy number of the 3S rRNA gene DNA is constant depending on the strain. In the present invention, it is possible to measure the abundance of a specific strain by performing the real-time quantitative PCR of the present invention using a complex microbial or symbiotic microbial homogenate. This method is also within the technical scope of the present invention.

[0103]

Next, the present invention will be described more specifically with reference to examples and comparative examples. Example 1 Synthesis of Nucleic Acid Probe: The base sequence of target nucleic acid A is composed of oligodeoxyribonucleotide (5 ′) tgc cat ccc ctc
The nucleic acid probe of the present invention was synthesized in the following order as aat gg (3 ′).

Design of Nucleic Acid Probe: Since the base sequence of the target nucleic acid is as described above, the base sequence of the nucleic acid probe can be simply composed of oligodeoxyribonucleotide (5 ′) cc.
Designed as a ttg agg gga tgg ca (3 '). The nucleic acid probe of the present invention was designed as follows. A donor dye BODIPY FL (Molecular Probes, US
A) is attached to the acceptor dye BODIPY 581/591 (Molecular
Probes, D-2228, USA) (BODIPY
FL- (5 ') cca (BODIPY 581/591) ttg agg gga tgg ca (3')
Design: Nucleic acid probe 1).

The 5'Amino-Modifier C6 kit (Glen Resear
ch Inc., a phosphate group thymidylate using USA) linker - modified with (CH _{2) 6} -SH. The OH group at position 6 of the base ring of thymidine was modified with a linker-(CH ₂ ) ₇ -NH ₂ using an Amino-Modifier C2dT kit (Glen Research, USA).
Using these modified thymidylates and thymidine, DN
An oligonucleotide having the following base sequence was synthesized using an A synthesizer (ABI394) (Perkin Elmer Japan Applied). That is, HS- (CH ₂ ) _6- (5 ′) cc
_{a (H 2 N- (CH 2} ) 7 -) ttg agg gga tgg ca ' in deoxyribooligonucleotide having the base sequence of, 5 (3)' phosphate group of terminal the linker - (CH _{2) 6} -SH And the OH group at the 6-position C of the base ring of the fourth thymine from the 5 'end is a linker-(CH
Are modified with _{2) 7} -NH _2. The synthesis of DNA is β-
Cyanoethylphosphamidite (2-cyanoethylphos
phoramidite) method with TrON at the 5 'end. After synthesis, elimination of the protecting groups was carried out at 55 ° C. and 5% with 28% aqueous ammonia.
Went on time.

Purification of synthesized product: The synthetic oligonucleotide obtained above was dried to obtain a dried product. 0.5M NaHCO
₃ / Na ₂ CO ₃ buffer (pH 9.0). NAP the lysate
Gel filtration was performed using a -10 column (manufactured by Pharmacia) to remove unreacted substances.

Labeling of acceptor dye: The filtrate was dried and dissolved in 150 μL of sterilized water (oligonucleotide
A solution). 1 mg BODIPY 581 / 591-NHS (Molecular Probes
USA, USA) was dissolved in 100 μL of DMF (dimethylformamide), and the oligonucleotide A solution, 1M NaHCO ₃ / Na ₂ C
150 μL of O ₃ buffer was added, and the mixture was stirred and reacted at room temperature overnight.

Purification of synthesized product: The reaction product was subjected to gel filtration with NAP-25 (manufactured by Pharmacia) to remove unreacted products. Then, the protecting group was eliminated with 2% TFA. Reverse-phase HPLC using a SEP-PAC _C18 column was performed, and the linker-(CH ₂ ) ₇ -NH ₂ of the oligonucleotide was used as an acceptor dye BODIPY 581 /
The target compound to which 591 was bound was fractionated. The fraction was subjected to gel filtration with NAP-10 (Pharmacia).

Labeling of Donor Dye: The gel filtrate was dried and dissolved in 150 μL of sterile water (oligonucleotide B).
solution). 1mg BODIPY FL-Chloride (Molecular Prob
es, USA) in 100 μL of DMF, add the above oligonucleotide B solution, 150 μL of 1 M NaHCO ₃ / Na ₂ CO ₃ buffer, stir, react at room temperature overnight, and react with the 5′-end linker.
The donor dye BODIPY FL was bound to-(CH ₂ ) ₆ -SH.

Purification of synthesized product: The reaction product was subjected to gel filtration with NAP-25 (manufactured by Pharmacia) to remove unreacted products. A nucleic acid of the present invention obtained by performing reverse phase HPLC in the same manner as described above, which is an oligonucleotide in which the acceptor dye BODIPY 581/591 is bound to the thymine base at the fourth position from the 5 ′ end, and the donor dye BODIPY FL is added to the 5 ′ end. A probe, that is, the nucleic acid probe 1 of the present invention labeled with a donor dye and an acceptor dye was obtained. The nucleic acid probe of the present invention was eluted later than the oligonucleotide to which the acceptor dye was bound.

The nucleic acid probe of the present invention was quantified by measuring a value at 260 nm with a spectrophotometer. The probe was measured for 650 using a spectrophotometer.
Scanning at ~ 220nm absorbance resulted in a BODI
It was confirmed that PY FL and BODIPY 581/591 had DNA absorption. Further, the purity of the purified product was determined by reverse phase HPLC as described above, and as a result, it was confirmed that the product was a single peak.

The conditions for the above reverse phase chromatography are as follows: Elution solvent A: 0.05N TEAA 5% CH ₃ CN Elution solvent B (for gradient): 0.
05N TEAA 40% CH ₃ CN column: SEP-PAK C18; 6 × 250 mm Elution rate: 1.0 ml / min Temperature: 40 ° C. Detection: 254 nm

Example 2 Probe Labeled Only with Acceptor Dye BODIPY 581/591
((5 ') cca (BODIPY 581/591) ttg agg gga tgg ca
(3 ′)) Synthesis of (Nucleic Acid Probe 2): Synthesis was performed in the same manner as for nucleic acid probe 1, except that the phosphate group at the 5 ′ end was labeled with donor dye BODIPY FL. Example 3 A probe labeled only with the donor dye BODIPY FL ((BODIPY
FL) (5 ') cca ttg agg ggatgg ca (3')) (Nucleic acid probe
Synthesis of 3): The acceptor dye BO is added to the 5'-terminal phosphate group.
Synthesis was performed in the same manner as in the nucleic acid probe 1 except that the operation of labeling with DIPY 581/591 was excluded.

Example 4 Synthesis of Target Nucleic Acid: An oligonucleotide having the nucleotide sequence of (5 ′) tgc cat ccc ctcaat gg (3 ′) was synthesized in the same manner as in the synthesis of the above oligonucleotide, and The target nucleic acid was used.

Example 5 An antibody obtained by hybridizing a probe of the present invention to a target nucleic acid was prepared.
Fluorescence intensity measurement: quartz cell (10 mm x 10 mm) (capacity 4 m
L) in 500 μL buffer (120 mM NaCl (final concentration 30 mM), 120 mM T
ris-HCl (final concentration 30 mM; pH = 7.2) was added, and then 1460 μL of sterile distilled water was added and stirred. 8.0 μL of the nucleic acid probe 1 or nucleic acid probe 2 (10 μM) solution of the present invention was added thereto, and the mixture was stirred (final concentration of nucleic acid probe: 40 nM). 30 ℃
The fluorescence wavelength was scanned while keeping the temperature (excitation wavelength: 490 nm)
(8 nm width)), measured fluorescence wavelength: 580 nm (8 nm width). Next, after controlling the reaction system to 60 ° C., 32.0 μL of a 10 μM target nucleic acid solution was added (final target nucleic acid concentration: 160 nM), followed by stirring. Then, the fluorescence wavelength was scanned. Then, set the temperature to 3
The nucleic acid probe was hybridized with the target nucleic acid at 0 ° C., and the fluorescence wavelength was scanned. The results are shown in Tables 1 and 2. In addition, as a constant temperature cell holder, a crystallization program low temperature circulator PCC-7000 manufactured by Tokyo Scientific Instruments Co., Ltd.
Was used to control the cell temperature (reaction temperature). Also,
The fluorescence was measured using a fluorimeter LS50B manufactured by PerkinElmer.

[0116]

[0117]

In Table 1, the acceptor dye BODIPY 5
The fluorescence intensity of 81/591 was measured. In Table 2, the fluorescence intensity of the donor dye was measured. From Table 1, it can be seen that the fluorescence intensity of the acceptor dye prior to hybridization was higher than that of nucleic acid probe 2+
It turns out that it is extremely higher than that of the experimental system of the target nucleic acid. This is because, in the nucleic acid probe 1, the donor dye BODIPY FL and the acceptor dye BODIPY 581 /
This is because the FRET phenomenon occurred between 591 and the acceptor dye emitted light. On the other hand, since the nucleic acid probe 2 has no donor dye in the molecule, no FRET phenomenon occurred and no fluorescence was emitted.

After hybridization, there was a large decrease in the experimental system of nucleic acid probe 1 + target nucleic acid. In the experimental system of the fluorescence intensity of the nucleic acid probe 2 + the target nucleic acid, there was almost no change. This is because the energy was transferred to the GC hydrogen bond because the donor dye approached the GC hydrogen bond due to the hybridization between the nucleic acid probe 1 and the target nucleic acid.

Table 2 shows that the fluorescence intensity of the donor dye before hybridization was extremely higher in the experimental system of nucleic acid probe 1 + target nucleic acid than in the experimental system of nucleic acid probe 2 + target nucleic acid. It turns out that it is high. This is because in the nucleic acid probe 1, the donor dye BODIPY FL and the acceptor dye BODIPY 581/59
This is due to the fact that the FRET phenomenon occurred between 1 and the energy of the donor dye was transferred to the acceptor dye. On the other hand, since the nucleic acid probe 3 does not have an acceptor dye in the molecule, the FRET phenomenon does not occur and fluorescence is emitted.

After hybridization, in the experimental system of nucleic acid probe 1 + target nucleic acid, there was only a slight decrease. This is because the energy was transferred from the acceptor dye to the GC hydrogen bond because the donor dye approached the GC hydrogen bond by hybridization to the target nucleic acid. On the other hand, in the case of the experimental system in which the fluorescence intensity of the nucleic acid probe 3 + the target nucleic acid was significantly reduced. This is because the energy used for light emission was transferred to the GC hydrogen bond because the donor dye approached the GC hydrogen bond by hybridization to the target nucleic acid. As can be seen from Tables 1 and 2, the nucleic acid probe of the present invention is designed such that when hybridized to a target nucleic acid, the fluorescence intensity of both the donor dye and the acceptor dye decreases.

Example 6 In the same manner as in Examples 1, 2, 3 and 4, the target nucleic acid B:
An oligodeoxyribonucleotide having a base sequence of (5 ′) aacgatgcca tggatttgg (3 ′) was synthesized. In the same manner as in the above example, X-rhodamine was labeled as an acceptor dye, and BODIPY FL was labeled as a donor dye, to prepare a nucleic acid probe 4 of the present invention which hybridizes to the target nucleic acid B. Nucleic acid probe 4 had the following structure: BODIPY
FL- (5 ') ccaaat (X-Rhodamine) ccat ggcatca tt (3').

In a measurement system in which target nucleic acids A and B are present,
The nucleic acid probes 1 and 4 of the present invention were added to
Nucleic acid probe 1 and target nucleic acid B and nucleic acid probe 4
Of fluorescence intensity of the reaction system in which was hybridized: 500 μL buffer (120 mM NaCl (final concentration: 30 mM), 120 mM Tris-H) in a quartz cell (10 mm × 10 mm) (capacity: 4 mL).
Cl (final concentration 30 mM; pH = 7.2) was added and then 146
0 μL of sterile distilled water was added and stirred. There, 8.0
μL of nucleic acid probe 1 and nucleic acid probe 4 of the present invention
(10 µM) solution (final concentration of nucleic acid probe 40n)
M) Stirred. The fluorescence intensity was measured while keeping the temperature at 45 ° C.
(Probe 1: fluorescence measurement wavelength, 580 nm (5 nm width);
Probe 4: measurement wavelength, 610 nm (5 nm width); excitation wavelength (common): 490 nm (8 nm width)). Then, for each concentration shown in Table 3, each solution of target nucleic acids A and B
0 μL was added and stirred. Then, the fluorescence intensity was measured. Table 3 shows the results. In addition, the same thermostat cell holder, fluorescence measuring device, and the like were used as in the above-described embodiment, and the method was performed in the same manner as in the above-described embodiment.

[0124]

As can be seen from Table 3, 580 nm and 6
The fluorescence intensity at 10 nm decreases after hybridization according to the concentration of the added target nucleic acid. In the case of the excitation light of 490 nm, the fluorescence intensity of 580 nm is that of the probe 1 of the present invention, and that of 610 nm is that of the probe 4 of the present invention. Thus, even if a plurality of target nucleic acids are present in the measurement system, it can be understood that a plurality of target nucleic acids can be simultaneously and simply measured by using the nucleic acid probe of the present invention and the nucleic acid measurement method using the same.

[0126]

As described above, the nucleic acid probe of the present invention is constituted, and when hybridized to a target nucleic acid,
Since the fluorescence intensity of the donor dye and the acceptor dye both decrease significantly before and after hybridization, the target nucleic acid can be measured in a very small amount, accurately, simply, and in a short time. The donor dye has an energy of G
Since it can be transferred to a C hydrogen bond, its type is limited. However, since the acceptor dye does not need to have such properties, various dyes may be used as long as they have the property of undergoing energy transfer. Is available. That means
In the present invention, nucleic acid probes that emit various fluorescent colors (different fluorescent wavelengths) with one donor dye (one excitation wavelength) can be manufactured. This simplifies the optical system for excitation, since a nucleic acid measuring apparatus for measuring various types of nucleic acids only needs to be provided with one type of excitation laser. That is, the design and manufacture of the nucleic acid measuring device are also facilitated. In addition, since various types of nucleic acid probes can be used even with a simple nucleic acid measuring device having only one wavelength of excitation laser, it is possible to simultaneously measure many types of target nucleic acids.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 33/58 A 33/58 C12N 15/00 ZNAA (72) Inventor Takahiro Kanakawa Tsukuba, Ibaraki 1-1-1-1, Higashi-shi, Independent Administrative Law, National Institute of Advanced Industrial Science and Technology, Tsukuba Center (72) Inventor Yoichi Kamagata 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan Independent Administrative Law, Tsukuba Center, National Institute of Advanced Industrial Science and Technology (72) Invention Person Masaki Torimura 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture Independent Administrative Law, National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor Shinya Kurata Inside 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environmental Engineering Co., Ltd. (72) Inventor Kazutaka Yamada 1-9-8 Higashi Kanda, Chiyoda-ku, Tokyo Environmental Engineering Co., Ltd. 72) Inventor Toyoichi Yokomaku F-term (reference) 2G043 AA01 BA16 CA03 DA02 EA01 FA03 FA06 GA07 GB07 GB21 KA02 KA05 KA09 LA01 NA01 2G045 CB01 CB17 1-9-8 Higashikanda, Chiyoda-ku, Tokyo CB21 DA12 DA13 DA14 FA29 FB02 FB07 FB12 GC15 4B024 AA11 CA09 HA14 4B063 QA01 QA13 QA18 QQ42 QQ52 QR32 QR56 QS34 QS36 QX02

Claims (16)

[Claims] 1. A plurality of fluorescent dyes which form at least one pair of a fluorescent dye pair causing a FRET phenomenon, ie, a fluorescent dye (donor dye) which can be a donor dye and a fluorescent dye (acceptor dye) which can be an acceptor dye. In a single-stranded nucleic acid probe labeled with a dye, when the probe hybridizes to a target nucleic acid, the base sequence is designed so that the fluorescence intensity of the acceptor dye is reduced, and the dye is labeled. A novel nucleic acid probe for nucleic acid measurement, characterized in that: 2. The novel nucleic acid probe for nucleic acid measurement according to claim 1, wherein the fluorescence intensity of both the donor dye and the acceptor dye decreases. 3. A fluorescent dye which can be a donor dye is BODIPY.
FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamine,
3. The novel nucleic acid probe for nucleic acid measurement according to claim 1, which is 6-TAMRA. 4. The novel nucleic acid probe for nucleic acid measurement according to claim 1, which has a pair of a dye pair of a donor dye and an acceptor dye. 5. The nucleic acid probe is labeled at its terminal with a donor dye, and when the nucleic acid probe hybridizes to the target nucleic acid at the terminal, one nucleotide from the terminal base of the target nucleic acid hybridized to the probe. The nucleic acid measurement according to any one of claims 1 to 4, wherein the base sequence of the probe is designed such that at least one base G (guanine) is present in the base sequence of the target nucleic acid at a distance of 3 to 3 bases. New nucleic acid probe for 6. When a nucleic acid probe is hybridized to a target nucleic acid, a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair in the donor dye-labeled portion. The novel nucleic acid probe for nucleic acid measurement according to any one of claims 1 to 5, wherein a base sequence of the probe is designed. 7. The novel nucleic acid probe for nucleic acid measurement according to any one of claims 1 to 6, wherein the donor dye labels the 5 ′ end (including the 5 ′ end) of the nucleic acid probe. 8. The novel nucleic acid probe for nucleic acid measurement according to claim 1, wherein the donor dye labels the 3 ′ end (including the 3 ′ end) of the nucleic acid probe. 9. The novel nucleic acid probe for nucleic acid measurement according to claim 7, wherein the 5 ′ terminal base of the nucleic acid probe is G or C and the 5 ′ terminal is labeled with a donor dye. 10. The novel nucleic acid probe for nucleic acid measurement according to claim 8, wherein the 3 ′ terminal base of the nucleic acid probe is G or C and the 3 ′ terminal is labeled with a donor dye. 11. The novel nucleic acid probe for nucleic acid measurement according to at least one of claims 1 to 10, which hybridizes to the target nucleic acid in a measurement system in which at least one or more target nucleic acids are present. A novel nucleic acid probe for nucleic acid measurement, and a novel nucleic acid probe for nucleic acid measurement having at least the same number of species as the target nucleic acid and having a different fluorescence color from the present nucleic acid. A novel method characterized in that a nucleic acid is hybridized and the amount of decrease in fluorescence intensity at the measurement wavelength before and after hybridization is measured at least as many wavelengths as the number of species of the novel nucleic acid probe for nucleic acid measurement. Nucleic acid measurement method. 12. A kit for measuring the concentration of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present, wherein the kit contains or accompanies the nucleic acid probe according to at least one of claims 1 to 10. Characteristic kit for nucleic acid measurement. 13. A method for analyzing or measuring polymorphism and / or mutation of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present, wherein at least one of the claims 1 to 10 is used. The novel nucleic acid probe for nucleic acid measurement according to the section, the number of species at least as many as the number of species of the target nucleic acid, and the presence of a novel nucleic acid probe for nucleic acid measurement of different fluorescent emission color in the measurement system, The target nucleic acid is hybridized with the novel nucleic acid probe for nucleic acid measurement, and the amount of decrease in the fluorescence intensity at the measurement wavelength before and after hybridization is at least as many as the number of species of the novel nucleic acid probe for nucleic acid measurement. A method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, wherein the method comprises: 14. A measurement kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid in a measurement system in which at least one or more target nucleic acids are present.
A measurement kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, comprising or accompanying the novel nucleic acid probe for nucleic acid measurement according to at least one of the above. 15. The nucleic acid measuring method according to claim 11, wherein the target nucleic acid is derived from a microorganism or animal obtained by pure isolation. 16. The nucleic acid measuring method according to claim 11, wherein the target nucleic acid is a nucleic acid contained in a cell of a complex microorganism system or a symbiotic microorganism system or in a homogenate of the cell.