JP2001286300A - Method for measuring nucleic acid, nucleic acid probe used therefor, and method for analyzing data obtained by the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid measuring method using a nucleic acid probe labeled with a fluorescent dye, and real-time quantitative PCR using the method
And a method for analyzing data obtained by the real-time quantitative PCR method in a short time and accurately. SOLUTION: A novel nucleic acid measuring method in which a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid and the amount of decrease in the emission of the fluorescent dye before and after hybridization is measured, and a real-time quantitative PCR method using the method And a data analysis method having a process of correcting the fluorescence intensity value during the annealing reaction with the value during the denaturation reaction when analyzing data obtained by the PCR method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a nucleic acid,
The present invention relates to a nucleic acid probe used for the method and a method for analyzing data obtained by the method. More specifically, the present invention relates to various methods for measuring various nucleic acids using a nucleic acid probe labeled with a fluorescent dye, and reduces the emission of the fluorescent dye before and after hybridization caused when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid. New measurement methods for various nucleic acids based on the principle of measuring the amount, nucleic acid probes and devices (de)
a method for analyzing data obtained by the PCR method, which is one of the measurement methods, an analysis device, and a computer-readable recording medium in which each means of the analysis method is recorded as a program.

[0002]

2. Description of the Related Art Conventionally, various methods for measuring the amount of a nucleic acid using a nucleic acid probe labeled with a fluorescent dye are known.
The method includes the following. (1) Dot blotting method In this method, after a target nucleic acid and the nucleic acid probe are hybridized on a membrane, unreacted nucleic acid probes are washed away, and a fluorescent dye molecule labeled on the nucleic acid probe hybridized with the target nucleic acid is used. Only the fluorescence intensity is measured.

(2) Intercalator method (Glazer et.
al., Nature 359: 959,1992): This method measures the amount of increase in luminescence because a certain fluorescent dye called an intercalator emits strong light when it gets stuck in the duplex of nucleic acid. How to As the fluorescent dye, for example, ethidium bromide (Experimental Medicine, Vol. 15, No. 7)
No. 46-51, 1997, Yodosha), SYBR R
Green I (LightCycler ^™ System; 1999)
Brochure issued by Roche Diagnostics Co., Ltd. on April 5, 2008).

(3) FRET (fluorescence energy transfe)
r) (Mergney et al., Nucleic Acid Re
s., 22: 920-928, 1994.): This method consists of two nucleic acid probes that hybridize to the target nucleic acid. Each of the two nucleic acid probes is labeled with a different fluorescent dye. One of the two probes is labeled with a fluorescent dye capable of transmitting energy to the fluorescent dye of the other probe and causing it to emit light through the FRET phenomenon.
The two probes are placed so that the fluorescent dyes face each other and
It is designed to hybridize 〜9 bases apart. Thus, when the two nucleic acid probes hybridize to the target nucleic acid, luminescence of the fluorescent dye occurs, and the degree of luminescence is proportional to the amount of the target nucleic acid replicated.

(4) Molecular beacon method (Tyagi et al.,
(Nature Biotech., 14: 303-308, 1996.) The nucleic acid probe used in this method has a reporter dye at one end of the nucleic acid probe and a quencher dye at the other end. And since both ends are complementary to each other in the base sequence,
The nucleotide sequence is designed so that the probe as a whole forms a hairpin stem. When suspended in a liquid due to its structure, the emission of the reporter dye is suppressed by the quencher dye due to Forster resonance energy. However, hybridization to the target nucleic acid breaks the hairpin stem structure, increasing the distance between the reporter dye and the quencher dye.
No transfer of resonance energy occurs. for that reason,
Emission of the reporter dye occurs.

(5) Davis method (Davis et al., Nu
cleic acids Res. 24: 702-706, 1996) Davis added a fluorescent dye at the 3 'end of
Probes bound via a specer with multiples were generated. This was applied to flow cytometry. It was reported that 10 times more fluorescence intensity was obtained when hybridization was performed than when a fluorescent dye was directly bonded to the 3 ′ end. These methods include various methods for measuring nucleic acids, HI
SH method (fluorescent in situhybridization assay
s), PCR method, LCR method (ligase chain reactio
n), SD method (strand displacement assays), competitive hybridizatio
n), etc., and it has achieved remarkable development.

[0007] These methods, which are currently generally used, require a nucleic acid probe labeled with a fluorescent dye to undergo a hybridization reaction, and then the unhybridized nucleic acid probe to be washed out of the reaction system. There is an unfavorable procedure.
Obviously, omitting such a procedure results in a reduction in measurement time, simplicity of measurement, and accuracy of measurement. Therefore, development of a nucleic acid measurement method that does not have such a procedure has been desired.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a method for measuring nucleic acid using a nucleic acid probe labeled with a fluorescent dye in a shorter time, more simply,
An object of the present invention is to provide a method capable of more accurately measuring the amount of a target nucleic acid.

[0009]

Means for Solving the Problems In order to solve the above problems, the present inventors have repeatedly studied a method for measuring a nucleic acid using a nucleic acid probe. As a result, when a nucleic acid probe labeled with a fluorescent dye hybridizes to a target nucleic acid, there is a phenomenon (fluorescence quenching phenomenon) in which the emission of the fluorescent dye decreases (fluorescence quenching phenomenon). In addition, the inventors have discovered that the degree of the reduction depends on the type or base sequence of the base to which the fluorescent dye is bound. The present invention has been completed based on such findings.

That is, the present invention provides 1) a nucleic acid measuring method using a nucleic acid probe labeled with a fluorescent dye, wherein when the nucleic acid probe hybridizes to a target nucleic acid, the fluorescent dye reduces its emission. Wherein the nucleic acid probe is hybridized to a target nucleic acid, and the amount of decrease in emission of the fluorescent dye before and after hybridization is measured. 2) A nucleic acid probe labeled with a fluorescent dye Is hybridized to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces its emission, and the probe is labeled with a fluorescent dye at its end, and the nucleic acid probe is attached to the target nucleic acid. Upon hybridization, the probe is From terminal base where the target nucleic acid hybridized 1
A nucleic acid probe for nucleic acid measurement, wherein the base sequence of the probe is designed so that G (guanine) is present in the base sequence of the target nucleic acid at least one or more bases at a distance of 3 to 3 bases; When a nucleic acid probe labeled with a dye hybridizes to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces its emission, and the probe is labeled with a fluorescent dye at its end, The nucleic acid probe,
The base sequence of the probe is designed such that, when hybridized to the target nucleic acid, the base pair of hybridization forms at least one pair of G (guanine) and C (cytosine) at the terminal portion. A nucleic acid probe for nucleic acid measurement,
Nucleic acid measurement method using those probes,

3) Polymorphism (poly) of the target nucleic acid or gene
In a method for analyzing or measuring morphism or / and mutation, the nucleic acid probe according to 2) above is hybridized to a target nucleic acid or gene, and the amount of change in fluorescence intensity is measured. Or 4) a method for analyzing or measuring a polymorphism or / and mutation of a gene, and 4) a measurement kit for analyzing or measuring a polymorphism or / and a mutation of a target nucleic acid or gene.
A measurement kit for analyzing or measuring a polymorphism or / and mutation of a target nucleic acid or gene, which comprises the nucleic acid probe according to

5) In the method for analyzing data obtained by the method described in 3) above, the fluorescence intensity value of the reaction system when the target nucleic acid or gene is hybridized with a nucleic acid probe labeled with a fluorescent dye, A data analysis method for a method for analyzing or measuring a polymorphism or / and mutation of a target nucleic acid or gene, characterized by having a process of correcting with a fluorescence intensity value of the reaction system when not hybridizing Also,

6) A measuring device for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid or gene, comprising a means for performing the data analysis method described in 5) above, A measuring device for analyzing or measuring a polymorphism and / or mutation of a gene; 7) a computer-readable recording medium on which a program for causing a computer to execute the correction process described in 5) above;

8) A nucleic acid measuring method using a probe having the nucleic acid probe described in 1) or 2) bonded to a solid surface, and a nucleic acid measuring device of the method; and 9) the above 1), 2) or HISH (fluorescent in situ hybridization assa) using the nucleic acid measurement method described in 8).
ys) method, also

10) A method for analyzing data obtained by the nucleic acid measurement method described in the above 1), 2) and 9), and 11) P using the nucleic acid measurement method described in the above 1) and 2).
CR method; 12) a data analysis method obtained by the PCR method described in 11) above; 13) a nucleic acid melting curve analysis method using the PCR method described in 11) above; And 13) an analysis method obtained by fusing; and 15) a PCR measurement and / or analysis device having a means for analyzing by the analysis method described in 11), 12), 13) or 14) above;

16) The above 11), 12), 13) or 1
A computer-readable recording medium in which a procedure for analysis by the analysis method according to 4) is recorded as a program, and 17) a nucleic acid characterized by utilizing the data analysis method according to 12) or 14). A quantification method; and 18) a nucleic acid measurement method using the PCR measurement and / or analysis device described in 15) above, and 19) a nucleic acid measurement using the recording medium described in 16) above. How to provide.

[0017]

Next, the present invention will be described in more detail with reference to preferred embodiments. In the present invention, DN
A, RNA, cDNA, mRNA, rRNA, XTP
s, dXTPs, 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, S
PC using tretch PCR, reverse PCR, Alu sequence
R, multiplex PCR, PCR using mixed primers, PN
A, PCR method and hybridization method (hy
bridization assays), HISH (fluorescent in situ
hybridization assays), PCR (polymerase c)
hain assays), LCR method (ligase chain reactio)
n), SD method (strand displacement assays), competitive hybridizat method
terms), DNA chips, nucleic acid detection (gene detection) devices, SNPs (snips: single nucleotide substitution polymorphisms), complex microbial systems, etc. are now commonly used in molecular biology, genetic engineering, microbial engineering, etc. Has the same meaning as the term used for A first feature of the present invention is a method for measuring a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye, wherein the decrease in the emission of the fluorescent dye before and after hybridization occurs when the nucleic acid probe hybridizes to the target nucleic acid. Is to measure the quantity.

In the present invention, the measurement of a target nucleic acid means quantitative or quantitative detection, or simple detection.
A nucleic acid measurement method using a nucleic acid probe labeled with a fluorescent dye is a hybridization method (hybridization a).
ssays), HISH method (fluorescent in situhybridiza
tion assays), PCR method (polymerase chain assay)
s), LCR method (ligase chain reaction), SD method (str
and displacement assays) and competitive hybridization. In these methods, after adding a nucleic acid probe labeled with a fluorescent dye, the unreacted fluorescent dye of the nucleic acid probe not hybridized to the target nucleic acid is removed from the measurement system by a method such as washing, and the target nucleic acid is added to the target nucleic acid. The fluorescent dye labeled on the hybridized nucleic acid probe is emitted directly from the probe or by applying an indirect means to the probe (for example, by the action of an enzyme) to emit light. Is a method of measuring
The present invention is characterized in that a target nucleic acid is measured without such complicated operations.

In the present invention, a target nucleic acid refers to a nucleic acid intended for quantitative or quantitative detection, or simple detection. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids may be mixed. For example,
Complex microbial system (RNA or gene DN of multiple microorganisms)
A) is a specific nucleic acid intended for quantitative or quantitative detection in a symbiotic microorganism system (mixed system of RNA or gene DNA of a plurality of animals and plants and / or a plurality of microorganisms) or simply detection. 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. Specific examples of the above nucleic acids include DNA, RNA,
PNA, 2-O-methyl (Me) RNA, deoxyribo-oligonucleotides,
Riboxy-origonucleotide
s) and the like.

In the present invention, a fluorescent dye generally used for labeling and detecting a nucleic acid can be conveniently used by labeling a nucleic acid probe. However, when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid, To
It is preferable that the fluorescent dye labeled on the probe reduce its emission. For example, fluorescein or derivatives thereof {for example, fluorescein isothiocyana
te) (FITC) or its derivatives, such as Alexa 488, Alexa
532, cy3, cy5, EDANS (5- (2'-aminoethyl) amino-1-na
phthalene sulfonic acid｝, rhodamine (rhodamin)
e) 6G (R6G) or a derivative thereof (eg, tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (tetramethylrhodami
ne isothiocyanate) (TMRITC), x-rhodamine (x-rhod
amine), Texas red, BODIP
Y) FL (trade name; Molecular Probe)
s), United States), BODIPY FL / C3 (trade name; Molecular Probes, USA), BODIPY FL / C6 (trade name; Molecular Probes) (USA), BODIPY5-FAM (trade name; molecular probe (M
olecular Probes), USA), BODIPY T
MR (trade name; Molecular Probe)
es), USA), or a derivative thereof (eg, BODIPY TR (trade name); Molecular Probe (Mole)
Molecular Probes), USA), BODIPY R6G
(Trade name: Molecular Probe
s), BODIPY 564 (trade name; Molecular Probes, U.S.A.), BODIPY 581 (trade name, molecular
Probe (Molecular Probes), USA). Among these, FITC, EDANS, 6-joe,
TMR, Alexa 488, Alexa 532, BODIPY FL / C
3 (Trade name: Molecular Probe)
s), BODIPY FL / C6 (trade name; Molecular Probes, USA) and the like, as well as FITC, TMR, 6
-jeo, BODIPY FL / C3 (trade name; Molecular Probes, USA), BODIPY FL / C6 (trade name; Molecular Probes, USA) Can be cited as more preferable.

[0021] The nucleic acid probe to be hybridized to the target nucleic acid may be composed of oligodeoxyribonucleotides or oligoribonucleotides. In addition, both of them intervene in chimeric oligonucleotides (chemiric oligonucleotides).
dite). In addition, 2-o-methyl oligoribonucleotides (2'-o-methyl oligoribonucleotides) (olig
5'-terminal nucleoside of oribonucleotide (nucleoside)
Side) part may be cytidine, and the OH group at the 2′-position of the cytidine is modified with a methyl group. Alternatively, the 2′-o-methyl oligoribonucleotide may be interposed in an oligodeoxynuclueotide in order to increase the affinity for RNA.

A nucleic acid probe in which a modified RNA such as 2'-o-methyl oligoribonucleotide is interposed in an oligodeoxynucleotide is mainly used for measuring RNA. RNA using the probe
Is measured, the RNA solution, which is a sample, is 80 to 100 before hybridization with the probe.
° C, preferably 90-100 ° C, optimally 93-97 ° C
It is preferable to destroy the higher-order structure of RNA by heat treatment for 1 to 15 minutes, preferably 2 to 10 minutes, and most preferably 3 to 7 minutes. The base chain of the nucleic acid probe is 3
When the number of bases is 5 or less, a helper probe such as (5 ′)
It is preferable to add an oligonucleotide having a nucleotide sequence of AGGCCGGCCCTTGACTTTCC T (3 ′) to the reaction solution. In this case, the helper probe may be an oligodeoxynucleotide form or a 2-o-methyl oligoribonucleotide form. However, when a nucleic acid probe having more than 35 base chains is used, it is only necessary to heat denature the target RNA. When such a nucleic acid probe of the present invention is hybridized to RNA, the fluorescence intensity decreases according to the amount of RNA in the reaction solution, and the final RNA concentration becomes about 150 pM.
RNA can be measured up to.

In the measurement of RNA by a conventional hybridization method using a nucleic acid probe, an oligodeoxynucleotide or oligoribonucleotide has been used as a nucleic acid probe. Since RNA itself has a strong higher-order structure, the hybridization efficiency between the probe and the target RNA is poor, and the quantitativeness is 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. As a result, in the method of the present invention, the hybridization efficiency is substantially 10%.
0%, and the quantitativeness is improved. In addition, it becomes much simpler than the conventional method.

The number of bases of the probe of the present invention is 5 to 50, preferably 10 to 25, particularly preferably 15 to 20.
It is. If it exceeds 50, the permeability of the cell membrane will be poor when used in the HISH 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 base sequence of the probe may be any as long as it specifically hybridizes to the target nucleic acid.
There is no particular limitation. Preferably, when a nucleic acid probe labeled with a fluorescent dye hybridizes to the target nucleic acid, (1) the base sequence of the target nucleic acid is separated from the terminal base by 1 to 3 bases from the terminal nucleic acid hybridized to the probe, and (2) The base sequence of the probe-nucleic acid hybrid complex at the terminal portion of the probe is G (guanine) and C (C) in which the base sequence of the probe is designed so that (guanine) is present in at least one base. It is preferable that the base sequence of the probe is designed so that at least one pair of (cytosine) is formed.

The oligonucleotide of the nucleic acid probe in the present invention can be produced by a general method for producing an oligonucleotide. For example, it can be produced by a chemical synthesis method, a microbial method using a plasmid vector, a phage vector or the like (Tetrahedron letters, Vol. 22, 1859-1862).
Page, 1981; Nucleic acids Research, Vol. 14, 6227-
6245, 1986). It is preferable to use a commercially available nucleic acid synthesizer (for example, ABI394 (Perkin
Elmer, USA)).

For labeling the oligonucleotide with a fluorescent dye, 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 this spacer. The thus synthesized oligonucleotide labeled with a fluorescent dye can be purified by reverse phase chromatography or the like to obtain a nucleic acid probe used in the present invention.

Also, a fluorescent dye can be bound to the 3 'end of the oligonucleotide. 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 Co., Ltd.).
mpany)). Further, by introducing a phosphate group, the phosphate group O
For example,-(CH ₂ ) _n -SH is introduced into the H group as a specifier. 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 with an amino group or SH group to this spacer. The thus synthesized oligonucleotide labeled with a fluorescent dye can be purified by reverse phase chromatography or the like to obtain a nucleic acid probe used in the present invention. When introducing this amino group, kit reagents (for example, Uni-link am
inomodifier (CLONTECH, USA), Fluo Reporter Kits F-6082, F-6083, F-6
084, F-10220 (Molecular probe (M
It is convenient to use Molecular Probes), USA)). Then, a fluorescent dye molecule can be bound to the oligoribonucleotide according to a conventional method. It is also possible to introduce a fluorescent dye molecule into the probe nucleic acid chain (ANALYTICAL BIOCHEMISTRY 225, pp. 32-38 (1998
Year)). Although the nucleic acid probe of the present invention can be prepared as described above, the preferred form of the probe is one in which the 3 ′ or 5 ′ end is labeled with a fluorescent dye, and the labeled base is G or C. It is something that is. When the 5 ′ end is labeled and the 3 ′ end is unlabeled, the 3 ′ terminal ribose or deoxyribose at the 3 ′ position C OH group is a phosphate group or the like, and the 3 ′ terminal ribose 2 ′ position C O
The H group may be modified with a phosphate group or the like without any limitation.

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 gene.
It can be suitably used for a method for analyzing or measuring sm) and / or mutation. In particular, the DN described below
A more convenient method is provided when used in combination with the A chip. That is, in the hybridization between the nucleic acid probe of the present invention and the target nucleic acid or gene, the fluorescence intensity changes depending on whether or not a GC pair is formed.
Then, the nucleic acid probe of the present invention is hybridized to the target nucleic acid or gene, and the luminescence intensity 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 or gene may be an amplified product obtained by one of various nucleic acid or gene amplification methods, or may be an extracted product. The target nucleic acid may be of any type. Examples of target nucleic acids to which the present invention can be applied include RNA, DNA, PNA, artificially modified nucleic acids, and the like. However, a guanine base may be present in the chain or at the end. If no guanine 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 (SNP)
Polymorphism analysis or the like can be analyzed or measured. At present, polymorphism analysis is currently performed by determining the nucleotide sequence of a nucleic acid or gene using the Maxam-Gilbert method and the dideoxy method.

The nucleic acid probe of the present invention is contained in a measurement kit for analyzing or measuring polymorphism and mutation of a target nucleic acid or / and a gene, thereby obtaining a polymorphism (po) of the target nucleic acid or a gene.
It can be suitably used as a measurement kit for analyzing or measuring lymorphism or / and mutation.

The polymorphism of the target gene of the present invention (polymorphis)
m) or / and mutation (mutation) when analyzing the data obtained by the method of analyzing or measuring, when the target nucleic acid is hybridized with a nucleic acid probe labeled with a fluorescent dye, the fluorescence intensity value of the reaction system, Providing a process of correcting the fluorescence intensity value of the reaction system when the above-mentioned ones are not hybridized makes the processed data highly reliable. Thus, the present invention provides a data analysis method for a method for analyzing or measuring polymorphism and / or mutation of a target nucleic acid or gene.

Further, a feature of the present invention is that polymorphism or / and mutation (mu)
tation), the fluorescence intensity value of the reaction system when the target nucleic acid or gene is hybridized with a nucleic acid probe labeled with a fluorescent dye, the reaction when the above-mentioned one is not hybridized. Having a processing means for correcting by the fluorescence intensity value of the system.
It is a measuring device for analyzing or measuring sm) or / and mutation.

Further, a feature of the present invention is that polymorphism and / or mutation (mu)
tation), the fluorescence intensity of the reaction system when the target nucleic acid or gene is hybridized with a nucleic acid probe labeled with a fluorescent dye, A computer-readable recording medium on which a program for causing a computer to execute a process of correcting with a fluorescence intensity value of a reaction system when the soybean is not soyed is recorded.

The probe of the present invention has a solid (supporting layer) surface,
For example, it may be fixed to the surface of a slide glass.
In this case, it is preferable that the end not labeled with the fluorescent dye is fixed. This format is now called a DNA chip. Monitoring of gene expression, determination of base sequence, mutation analysis (mu
tation analysis), single nucleotide polymorphism (single nucleotide p
olymorphism (SNP))
alysis). Of course, it can be used as a nucleic acid measurement device (chip).

For binding the 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 has been introduced, or an amino group can be introduced. First, a prepared slide glass is prepared. Then, i) react the phosphate group of the probe on the slide glass coated with the polycation, ii) react the probe with the amino group on the slide glass into which the aldehyde group is introduced, iii) react the amino group with the probe. PDC (pyridinium dichromat)
e) introduction, reaction with a probe having an amino group or an aldehyde group introduced, etc. (Fodor, PA, et a
l., Science, 251, 767-773 (1991); Schena, M., et al., Pro.
c. Natl. Acad. Sci., USA, 93, 10614-10619 (1996); McGa
l, G., et al., Proc.Natl.Acad.Sci., USA, 93,13555-13
560 (1996); Blanchard, AP, et al., Biosens. Bioelectro.
n., 11, 687-690 (1996)).

A device in which nucleic acid probes are arranged and bound in an array on a solid surface is more convenient for nucleic acid measurement. In this case, a large number of probes can be detected and quantified at the same time by making a device by binding many probes of the present invention having different base sequences individually on the same solid surface.
In this device, for each probe, at least one temperature sensor and a heater are provided on the surface opposite to the side where the solid probe is connected, and the temperature is set so that the region of the solid to which the probe is connected has an optimum temperature condition. Preferably, it is designed to be adjustable.

In this device, a probe other than the probe of the present invention, for example, two different fluorescent dyes in one molecule, and when not hybridized to the target nucleic acid, the two fluorescent dyes A nucleic acid probe having a structure that is quenched or luminescent by action but designed to fluoresce or quench when hybridized to a target nucleic acid, that is, the molecular beacon (Tyagi et al., Nature Biotech., 14: 303-308,199
The combination of 6) and the like can also be suitably used. Therefore, such a device is also included in the technical scope of the present invention.

The basic operation of the measuring method using the device of the present invention is as follows.
A solution containing a target nucleic acid such as A, cDNA, rRNA or the like is simply placed and hybridized. As a result, a change in the amount of fluorescence occurs in accordance with the amount of the target nucleic acid, and detection and quantification of the target nucleic acid can be performed from the amount of change in the fluorescence. In addition, by binding a large number of nucleic acid probes having different base sequences on one solid surface, it is possible to detect and quantify many nucleic acids at a time. Therefore, it is a novel DNA chip because it can be used 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 wash unreacted nucleic acids. Furthermore, by controlling the temperature for each type of nucleic acid probe with a micro heater, it is possible to control the optimal reaction conditions for each probe, so that accurate quantification becomes possible. In addition, the dissociation curve between the nucleic acid probe and the target nucleic acid can be analyzed by continuously changing the temperature with a minute 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 a target nucleic acid binds (fixes) a nucleic acid probe not modified with a fluorescent dye to a solid surface, hybridizes the target nucleic acid labeled with the fluorescent dye, and then performs hybridization. Unwashed target nucleic acids were washed away, and the fluorescence intensity of the remaining fluorescent dye was measured. To label a target nucleic acid with a fluorescent dye, for example, when targeting a specific mRNA, take the following steps: (1) m extracted from cells
Extract all RNA. (2) Then, using reverse transcriptase, cDNA is synthesized while incorporating a nucleoside modified with a fluorescent dye. Such an operation is not required in the present invention.

Although various probes are spotted on the device, the optimal hybridization conditions, such as temperature, for nucleic acids that hybridize to each probe are different. Therefore, the hybridization reaction and the washing operation must be performed under optimum conditions for each probe (for each spot). However, since it is physically impossible, hybridization is performed at the same temperature for all probes,
Cleaning is also performed at the same temperature and with the same cleaning liquid. Therefore, there have been disadvantages that the nucleic acid expected to be hybridized does not hybridize, and even if hybridized, the hybridization is not strong and is easily washed away. Quantitative properties of nucleic acids were low for such reasons. The present invention does not have such a disadvantage because the washing operation is not required. In addition, by providing a small 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. Therefore, in the present invention, the quantitativeness is significantly improved.

In the present invention, by using the above-described nucleic acid probe or device, a target nucleic acid can be
It can be measured simply and specifically. The measurement method is described below. In the measurement method of the present invention, first, the above-described nucleic acid probe is added to the measurement system and hybridized with the target nucleic acid. The method can be carried out by a commonly known method (Analytical Biochemistry, 183,
231-244, 1989; Nature Biotechnology, 14,
303-308, 1996; Applied and Environmental Mi
crobiology, 63, 1143-1147, 1997). For example, the hybridization conditions are as follows:
Molarity, preferably 0.1-1.0 molarity, pH is 6-8, preferably 6.5-7.5.

The reaction temperature is preferably within the range of the Tm value of the hybrid complex obtained by hybridization of the nucleic acid probe to a specific site of the target nucleic acid ± 10 ° C. By doing so, non-specific hybridization can be prevented. Tm-10 ° C
If it is less than Tm, nonspecific hybridization occurs. If it exceeds Tm + 10 ° C., no hybridization occurs. The Tm value can be determined in the same manner as in an experiment necessary for designing a nucleic acid probe used in the present invention. That is, an oligonucleotide having a complementary base sequence that hybridizes with the nucleic acid probe is chemically synthesized by the above-described nucleic acid synthesizer or the like, and the Tm value of a product hybridized with the nucleic acid probe is measured by a usual method.

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, in the present invention, the nucleic acid probe is hybridized to the target nucleic acid.
Then, before and after hybridization, the amount of emission of the fluorescent dye is measured with a fluorometer, and the amount of decrease in emission is calculated. Since the magnitude of the decrease is proportional to the amount of the target nucleic acid,
The amount 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 probe in the reaction solution is preferably 1.0 to 25.0 nM. When preparing a calibration curve, a probe is used for 1.
It is desirable to use at a ratio of 0 to 2.5.

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. Then, a plurality of concentrations of the probe are added, and the decrease in the fluorescence intensity value is measured. Then, the probe concentration that maximizes the decrease value of the measured 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 value of the fluorescence intensity measured with the probe at the preferable concentration.

The principle of the nucleic acid measuring method of the present invention is as described above, but various nucleic acid measuring methods such as FISH method, PCR method, LCR method, SD method, competitive hybridization method, TAS method, etc. Applicable to

An example is described below. a) When applied to the FISH method That is, the present invention is a method in which various kinds of microorganisms or other animals or plants are mixed and cannot be isolated from each other in cells of a microorganism system (complex microorganism system, symbiotic microorganism system) or It can be suitably applied to measurement of nucleic acids such as cell homogenates. The microorganism here is a microorganism generally referred to, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms,
Other examples include mycoplasma, virus, and rickettsie. The nucleic acid having a specific sequence is, for example, a nucleic acid having a base sequence having specificity for cells of a bacterial strain to be examined for how it is active in these microorganism systems. For example, the specific strain 5
Specific sequence of SrRNA, 16S rRNA or 23S rRNA or their gene DNA.

In the present invention, a nucleic acid probe is added to a complex microorganism system or a symbiotic microorganism system, and 5SrRN of a specific strain is added.
By measuring the amount of A, 16S rRNA or 23S rRNA or their gene DNA, the abundance of the specific strain in the system can be measured. In addition, a method of adding the nucleic acid probe to a complex microbial or symbiotic microbial homogenate, measuring the amount of decrease in the emission of a fluorescent dye before and after hybridization, and measuring the abundance of a specific strain is also a technique of the present invention. Within the target range.

The above measuring method is, for example, as follows.
is there. That is, before adding the nucleic acid probe,
Temperature, salt concentration and pH of the system or symbiotic microbial system
Adjust to Special features in complex or symbiotic microbial systems
The fixed strain has a cell number of 10 ⁷-10¹²Pcs / ml, preferred
Or 10⁹-10^TenIt is preferable to adjust to
is there. It is performed by dilution or concentration by centrifugation etc.
be able to. 10 cells⁷If the number is less than
The fluorescence intensity is weak and the measurement error increases. 10¹²Pieces / m
l, a complex or symbiotic microbial
Because the light intensity is too strong, the amount of specific microorganisms
It cannot be measured.

The concentration of the nucleic acid probe to be added depends on the number of cells of a specific strain in a complex microorganism system or a symbiotic microorganism system. 0.1 to 10 for a cell count of 1 × 10 ⁸ / ml.
The concentration is 0 nM, preferably 0.5 to 5 nM, more preferably 1.0 nM. When the concentration is less than 0.1 nM, the data does not accurately reflect the abundance of the specific microorganism. However, the optimal amount of the nucleic acid probe cannot always be determined 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 for hybridization to rRNA or its gene DNA is the same as that described above. Further, the reaction time is the same as the above conditions.
Under the conditions described above, the nucleic acid probe was
Hybridization is performed with NA, 16S rRNA or 23S rRNA or its gene DNA, and the decrease in luminescence of the fluorescent dye of the complex microorganism or 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, 1
This is because the amount of 6S rRNA or 23S rRNA or their gene DNA is proportional to the amount of the specific strain.

In the present invention, components other than microorganisms in the complex microbial system or the symbiotic microbial system are used as long as they do not inhibit the hybridization between the nucleic acid probe of the present invention and 5S rRNA, 16S rRNA or 23S rRNA of a specific strain or their gene DNA. There is no particular limitation as long as the emission of the fluorescent dye of the nucleic acid probe is not inhibited. For example, KH ₂ PO ₄ , K ₂ HPO ₄ , NaH ₂ PO ₄ , Na ₂ HPO ₄
Phosphates, ammonium sulfate, ammonium nitrate, inorganic nitrogens such as urea, various salts of metal ions such as magnesium, sodium, potassium, calcium ions, manganese, zinc, iron, sulfates of trace metal ions such as cobalt ions, Various salts such as hydrochloride and carbonate, and vitamins and the like may be appropriately contained. If the above-mentioned inhibition is observed, the cells may be separated from a bacterial system in which a plurality of microorganisms are mixed by an operation such as centrifugation, and suspended again in a buffer system 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. Its concentration depends on the type of buffer. The pH of the buffer is 4-12, preferably 5-9.

B) When applied to PCR methods 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, a specific nucleic acid probe of the present invention
R is performed, and the decrease in the emission of the fluorescent dye before and after the reaction is measured in real time. The PCR of the present invention means PCR by various methods. For example, RT-PC
R, RNA-primed PCR, Stretch PCR, reverse PC
It also includes PCR using R and Alu sequences, multiplex PCR, PCR using mixed primers, PCR using PNA, and the like. The term “quantitative” means quantitative measurement of the degree of detection in addition to the original quantitative measurement.

As described above, the target nucleic acid refers to a nucleic acid whose amount is quantitatively or quantitatively detected or simply detected. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids may be mixed. For example,
Complex microbial system (RNA or gene DN of multiple microorganisms)
A) or a symbiotic microbial system (mixed system of RNA or gene DNA of multiple animals and plants and / or multiple microorganisms)
Is the specific nucleic acid to be amplified. 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,
In the presence of Mg ions using dGTP, dCTP, dTTP or dUTP, target nucleic acid (DNA or RNA), Taq polymerase, primer, and nucleic acid probe or intercalator labeled with a fluorescent dye,
The target nucleic acid is amplified while the temperature is repeatedly reduced and increased, and the increase in the emission of the fluorescent dye in the amplification process is monitored in real time (Experimental Medicine, Vol. 15, No. 7, No. 4,
6-51, 1997, Yodosha).

The quantitative PCR method of the present invention is characterized by amplifying a target nucleic acid using the nucleic acid probe of the present invention, and measuring the amount of decrease in the emission of a fluorescent dye during the amplification process. In the quantitative PCR of the present invention, a preferable probe of the present invention has a base number of 5 to 50.
, Preferably 10 to 25, particularly preferably 15 to
At 20, any can be hybridized with the amplification product of the target nucleic acid during the PCR cycle. Further, it may be designed as either a forward type or a reverse type.

For example, the following can be mentioned. (1) The 5 'end, preferably the 5' end, is labeled with a fluorescent dye useful in the practice of the present invention, and when hybridized to the target nucleic acid at the end, the probe and the target nucleic acid are hybridized. The base sequence is designed so that G (guanine) is present in the base sequence of the target nucleic acid at least one base at least 1 to 3 bases away from the base portion at the 5 'end. (2) Among the probes of the above (1), a probe whose 3 ′ end is labeled with a fluorescent dye.

(3) Among the probes of the above (1), those in which the OH group at the 3′-terminal, for example, the 3′-terminal C of ribose or deoxyribose at the 3′-terminal is modified with a phosphate group, or , Wherein the OH group at the 2'-position C of ribose at the 3 'end is modified with a phosphate group or the like. (4) 3'-terminal, preferably 3'-terminal, which is labeled with the fluorescent dye of the present invention, and whose 3'-terminal base is G or C, or 2'-OH group of 3'-terminal ribose Is modified with a phosphate group or the like. (5) The probe of the above (1), wherein the 5 'end, preferably the 5' end, is labeled with the fluorescent dye of the present invention. (6) The 5 'end, preferably the 5' end, is labeled with a fluorescent dye useful in the practice of the present invention, and the 5 'end base is G or C.

In the cases (4) and (6), if the 5 ′ end cannot be designed to G or C from the base sequence of the target nucleic acid, the oligonucleotide 5 which is a primer designed from the base sequence of the target nucleic acid cannot be used. Even if 5'-guanylic acid or 5'-cytidylic acid is added to the 'terminal, 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 means, in addition to a probe designed from the base sequence of a target nucleic acid, 5 ′ at the 3 ′ or 5 ′ end of the probe. -Guanylic acid or 5'-cytidylic acid.

In particular, the probe (1), (2), (3) or (4) is designed so as not to be used as a primer. PCR is performed using one 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 degraded by the polymerase,
It is decomposed and removed from the hybridization product. 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. Also, the fluorescence intensity of a reaction system in which the target nucleic acid or the amplified target nucleic acid is hybridized with the probe (reaction system during annealing reaction or nucleic acid extension reaction until the probe is removed from the hybridization product by polymerase). Measure the value. Then, the amplified nucleic acid is measured by calculating the reduction rate of the fluorescence intensity value from the former. Fluorescence intensity when the probe is completely dissociated from the target nucleic acid or amplified target nucleic acid by a nucleic acid denaturation reaction or decomposed and removed from a hybridized product of the probe and the target nucleic acid or amplified target nucleic acid by polymerase during nucleic acid extension. The value is large. However, the probe is sufficiently hybridized to the target nucleic acid or the amplified target nucleic acid. The fluorescence intensity value of the reaction system before decomposition and removal is smaller than the former. The decrease in the fluorescence intensity value is proportional to the amount of the amplified nucleic acid. In this case, (2) such that the Tm of the hybridized product when the probe hybridizes with the target nucleic acid is in the range of ± 15 ° C., preferably ± 5 ° C. of the Tm value of the hybridized product of the primer. , (3) and (4) are desirably designed. If the Tm value of the probe is less than the Tm value of the primer, −5 ° C., especially −15 ° C., the fluorescence of the fluorescent dye does not decrease because the probe does not hybridize. Conversely, the primer Tm
If the value exceeds + 5 ° C., especially + 15 ° C., the probe will hybridize with the target nucleic acid which is not intended.
The specificity of the probe is lost.

The probes (5) and (6) are added as primers to a PCR reaction system. A PCR method using a primer labeled with a fluorescent dye has not yet been known except for the present invention. As the PCR reaction proceeds, the amplified nucleic acid is labeled with the fluorescent dye of the present invention.
Thus, 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 completed, the fluorescence intensity of the reaction system is lower than the former fluorescence intensity. .

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 having a low Mg ion concentration (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 Tm value can be determined by performing the PCR of the present invention and analyzing the nucleic acid melting curve of the amplified product. This method is a novel method for analyzing a melting curve of a nucleic acid. In this method, the nucleic acid probe used in the PCR method of the present invention or the nucleic acid probe used as a primer can be suitably used. In this case, by making the base sequence of the probe of the present invention a sequence complementary to the region containing SNP (snip; single nucleotide substitution polymorphism), the dissociation curve of the nucleic acid from the probe of the present invention after PCR is completed. By performing the analysis, the SNP can be detected from the difference between the dissociation curves. If the nucleotide sequence complementary to the sequence containing the SNP is used as the sequence of the probe of the present invention, the Tm value obtained from the dissociation curve between the probe sequence and the sequence containing the SNP indicates the dissociation between the probe sequence and the sequence containing no SNP. It is higher than the Tm value obtained from the curve.

The second feature of the present invention is the invention of a method for analyzing data obtained by the above-mentioned real-time quantitative PCR method. The real-time quantitative PCR method is currently a reaction device for performing PCR, a device for detecting the emission of a fluorescent dye, a user interface, that is, a computer-readable recording medium on which each procedure of the data analysis method is programmed and recorded. (Also known as Sequence Det
Section Software System) and a device consisting of a computer that controls them and analyzes the data, and is measured in real time. Thus, the measurement according to the invention is also performed with such a device.

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 Nucleotide Sequence Detection System (Sequence Detection S
ystem SDS 7700) (Perkin Elmer Applied Biosytems, Inc.
USA)) and LightCycler ^™ system (Roche Diagnostics, Germany).

The above-mentioned 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, the reaction can be repeatedly performed at a temperature of 95 ° C., 60 ° C., and 72 ° C.). It is. The detection system includes an argon laser for fluorescence excitation, a spectrograph, and a CCD camera. Further, each means of the data analysis method is programmed, and a computer-readable recording medium recording the program is installed in the computer, controls the above system via the computer, and outputs the data output from the detection system. This is a program that records a program for analyzing the data.

The data analysis program recorded on the computer-readable recording medium is a program for measuring the fluorescence intensity for each cycle, and using the measured fluorescence intensity as a function of the cycle to display a PCR amplification plot on a computer display. In the process shown above, the PCR cycle number (threshold cyclenum) at which the fluorescence intensity starts to be detected
ber: Ct), a calibration curve for obtaining the copy number of the sample nucleic acid from the Ct value, and a process of printing data and plot values of each process. PC
When R progresses exponentially, a linear relationship holds between the Log value of the copy number of the target nucleic acid at the start of PCR and Ct. Therefore, a calibration curve is created using a known number of copy numbers of the target nucleic acid, and the Ct of the sample containing the unknown copy number of the target nucleic acid is detected.
You can calculate the number of copies at the start.

The PCR-related invention of 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 an amplified nucleic acid in each cycle binds to a fluorescent dye, or when the amplified nucleic acid is a nucleic acid probe labeled with a fluorescent dye, An arithmetic processing step of correcting the fluorescence intensity value of the reaction system at the time of hybridization, by the fluorescence intensity value of the reaction system at the time of the above-mentioned combined or dissociated hybridized product in each cycle, that is, correction This is an arithmetic processing process.

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 labeled with the fluorescent dye" is specifically exemplified by each cycle of PCR. 40 ~
A reaction system at the time of nucleic acid extension reaction or annealing having a reaction temperature of 85 ° C, preferably 50 to 80 ° C can be mentioned. The specific temperature depends on the length of the amplified nucleic acid. The “reaction system when the bound or hybridized product is dissociated” is a reaction system for heat denaturation of nucleic acid in each cycle of PCR, specifically, for example, a reaction temperature of 90. -100 ° C, preferably 94-96 ° C.

The correction calculation process in the correction calculation process can be any process as long as it meets the object of the present invention. Specifically, the following formula (1) or (formula 2) 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 when the amplified nucleic acid binds to the fluorescent dye or when the amplified nucleic acid hybridizes to the nucleic acid probe labeled with the fluorescent dye in n cycles The fluorescence intensity value of the system, f _{den, n} : the combination in n cycles,
Alternatively, the fluorescence intensity of the reaction system when the hybridized product is dissociated]. Note that this process includes a process of plotting the corrected operation processing value obtained in this process or the value on a graph for each cycle number and displaying and / or printing it on a computer display.

The second feature is that the correction operation processing 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 each sample 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 : calculated by [Equation 3] or [Equation 4] in n cycles. fluorescent change rate or fluorescence change rate, f _n: correction processing value by [equation 1] or [equation 2] f _a: correction processing value by [equation 1] or [equation 2], the change in f _n is Any number of cycles before observation, but usually, for example, 10 to 40 cycles, preferably 15 to 30 cycles, more preferably 20 to 30 cycles You. ]. This process includes a process of plotting the calculated value, the comparison value, or the value obtained in the process on a graph for each cycle number, and displaying and / or printing the same on a computer display. , [Equation 1] or [Equation 2] may or may not include those steps.

The third feature is that 31) using the fluorescence change rate or the data of the fluorescence change rate calculated by [Equation 3] or [Equation 4],
The process of performing the arithmetic processing by [Equation 5], [Equation 6] or [Equation 7], log _b (F _n ), ln (F _n ) [Equation 5] log _b {(1-F _n ) × A}, ln {(1-F _n ) × b} [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} indicates a percentage (%). F _n : the rate of change of fluorescence or the rate of change of fluorescence in n cycles calculated by [Equation 3] or [Equation 4] 32) An operation processing step of calculating the number of cycles in which the operation processing value of the above 31) has reached a constant value 33) an arithmetic processing step of calculating the relational expression between the cycle number in a nucleic acid sample of known concentration and the copy number of the target nucleic acid at the start of the reaction, 34) an arithmetic processing step of calculating the copy number of the target nucleic acid in an unknown sample at the start of the PCR And a data analysis method. Then, a process consisting of 31) → 32) → 33) → 34) is preferred.

In each of the steps 31) to 33),
The method may include a step of plotting the operation processing value obtained in each step or the value on a graph with respect to each cycle number and displaying and / or printing on a computer display. The operation processing value in the process 34) is the same as described above, but includes at least the process since it needs to be printed. The corrected calculation processing value according to [Equation 1] or [Equation 2] and the calculation processing value according to [Equation 3] or [Equation 4] are plotted on a graph for each cycle number and displayed on a computer display. And / or may or may not be printed, so those steps may be added as needed.

The data analysis method is particularly effective when the real-time quantitative PCR method measures the amount of decrease in the emission of a fluorescent dye. A specific example is the above-described 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 feature is that real-time quantitative PCR
The respective means of the data analysis method for analyzing PCR using the analyzer of the present invention are programmed, and the computer executes the respective means 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 that, in the nucleic acid measurement method, the data analysis method, measurement and / or analysis apparatus of the present invention,
This is a novel nucleic acid measurement method using a recording medium.

The seventh 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. is there. That is, for the nucleic acid amplified by the PCR method of the present invention, a step of gradually increasing the temperature from a low temperature until the nucleic acid is completely denatured (for example, 50 ° C.)
To 95 ° C.), a process of measuring the fluorescence intensity at short time intervals (for example, 0.2 ° C. to 0.5 ° C.), a process of displaying the measurement results on a display every time, A process of displaying a melting curve of a nucleic acid, a process of first-order differentiation of the melting curve, and a value (−dF / dT, F:
This is an analysis method that includes a process of displaying on the display a differential value of fluorescence intensity (T: time) on a display 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, at the time of the nucleic acid extension reaction in each cycle, preferably, by adding to the above-mentioned process, a process 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, More favorable results are obtained.

In the present invention, the method for analyzing real-time quantitative PCR of the present invention, which is obtained by adding a method for analyzing a melting curve of a nucleic acid of the present invention to the above-mentioned data analysis method of the novel PCR method of the present invention, An analysis device is also within the technical scope of the present invention.

Further, one of the features of the present invention is a computer-readable recording medium on which a program for causing a computer to execute each means of the method for analyzing a melting curve of a nucleic acid of the present invention is recorded. In addition, the melting curve of the nucleic acid of the present invention is analyzed on a computer-readable recording medium that stores a program that enables a computer to execute each means of the data analysis method of the PCR method of the present invention. A computer-readable recording medium in which a program that enables a computer to execute each means of the method is additionally recorded.

The data analysis method, apparatus, and recording medium of the present invention are applicable to various fields such as medicine, forensic medicine, anthropology, ancient biology, biology, genetic engineering, molecular biology, agriculture, and plant breeding. Available at Further, it can be suitably used for a complex microbial system, a symbiotic microbial system, and a microbial system in which various kinds of microorganisms or other animals and plants are mixed and cannot be isolated from each other. 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, by using the data analysis method, apparatus and recording medium of the present invention, the copy number of 5S rRNA, 16S rRNA or 23S rRNA of a specific strain or their gene DNA in a complex microorganism system or a symbiotic microorganism system is determined. The amount of the specific strain in the system can be measured. It is 5S rRNA,
Genetic DNA of 16S rRNA or 23S rRNA
Is constant depending on the strain. In the present invention, the real-time quantitative PCR of the present invention is performed using a complex microbial or symbiotic microbial homogenate.
And the method for measuring the abundance of the specific strain is also within the technical scope of the present invention.

[0087]

Next, the present invention will be described more specifically with reference to examples and comparative examples. Example 1 Hybridization to the nucleobase sequence of 16S rRNA of Escherichia coli, ie, (3 ') CCGC
A nucleic acid probe having the nucleotide sequence of TCACGCATC (5 ′) was prepared as follows.

Preparation of nucleic acid probe : (3 ′) CCGCTCACGCATC
A deoxyribooligonucleotide having a base sequence of (5 ′), in which — (CH ₂ ) ₇ —NH ₂ is bonded to the OH group at the 3′-position carbon of deoxyribose at the 3 ′ end, is obtained from Medland Certified. The Resin Company (Mi
dland Certified Reagent Company, USA). In addition, Molecular Probes
From Fluo Reporter Kit
s) F-6082 (BODIPY FL propionic acid succinimidyl ester of BODIPY FL)
kit containing a reagent that binds the compound to the amine derivative of the oligonucleotide in addition to the ester). The kit was allowed to act on the purchased oligonucleotide to synthesize a nucleic acid probe labeled with bodypi FL used in the present invention.

Purification of synthesized product : The obtained synthesized product was dried to obtain a dried product. 0.5M NaHCO ₃ / Na ₂ CO
_It was dissolved in ₃ buffers (pH 9.0). The lysate is converted to NA
Gel filtration was performed using a P-25 column (manufactured by Pharmacia) to remove unreacted substances. In addition, reverse phase HPLC (B gradient:
15-65% for 25 minutes) under the following conditions. Then, the eluting main peak was collected. The fractionated fraction was freeze-dried to obtain a nucleic acid probe in a yield of 23% from 2 mM of the initial oligonucleotide raw material.

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

Example 2 50 ml of sterilized nutrient broth (NB) (manufactured by Difco) liquid medium (composition: NB, 0.08 g / 10
E. coli JM109 strain was cultured with shaking at 37 ° C. overnight using a 200 ml Erlenmeyer flask containing 0 ml).
Next, an equivalent amount of 99.7% ethanol was added to the main culture solution. 2 ml of the ethanol-added culture was centrifuged in a 2.0 ml Eppendorf centrifuge tube to obtain bacterial cells. 30 mM phosphate buffer (soda salt) (pH: 7.
2) The cells were washed once with 100 μl. 130 m of cells
The suspension was suspended in 100 μl of the above phosphate buffer containing M NaCl. The suspension was sonicated for 40 minutes in ice cooling (output: 33 w, oscillation frequency: 20 kHz, oscillation method: 0.5
Second oscillation, 0.5 second pause) to produce a homogenate.

After the homogenate was centrifuged, the supernatant was collected and transferred to a cell of a fluorometer. It was adjusted to 36 ° C. Then, a solution of the nucleic acid probe previously heated to 36 ° C. was added to a final concentration of 5 nM. Three
Escherichia coli 16S rRNA was hybridized with the nucleic acid probe for 90 minutes while controlling the temperature at 6 ° C. Then, the emission amount of the fluorescent dye was measured with a fluorimeter.

The amount of luminescence of the fluorescent dye before hybridization was determined by using 130 mM NaCl instead of the above supernatant.
30 mM phosphate buffer (soda salt) (pH:
The value measured using 7.2) was adopted. The emission amount was measured by changing the ratio of the amount of the nucleic acid probe to the amount of the supernatant (excitation light: 503 nm; measured fluorescence color: 512 nm). The result is shown in FIG. As can be seen from FIG. 1, the emission of the fluorescent dye decreases as the ratio of the supernatant increases. That is, in the present invention, it can be seen that the magnitude of the decrease in the emission of the fluorescent dye increases in proportion to the amount of the target nucleic acid hybridized to the nucleic acid probe.

Example 3 Preparation of nucleic acid probe: 23SrR of E. coli JM109 strain
(5 ') CCCACATCGTTTTG that hybridizes to NA
5 'of the oligonucleotide having the nucleotide sequence of TCTGGG (3')
-(CH ₂ ) is added to the OH group at the 3′-position carbon of the terminal nucleotide.
_7- NH ₂ was combined with Medland Certified Resin Company (MiMi) in the same manner as in Example 1.
dland Certified Reagent Company, USA). Further, the molecular probe (Mo
lecular Probes) from Fluor Reporter Kit (Fl
uoReporter Kit) F-6082 (BODIPY FL propionic acid su
In addition to ccinimidyl ester), a kit containing a reagent that binds the compound to an amine derivative of an oligonucleotide was purchased. The kit was allowed to act on the oligonucleotide to synthesize a nucleic acid probe labeled with bodypy FL. The obtained synthesized product was purified in the same manner as in Example 1 to obtain a nucleic acid probe labeled with Bodepy FL in a yield of 25% from 2 mM of the initial oligonucleotide raw material.

Example 4 A strain of Pseudomonaspus mobilis 421Y (Pseudomonaspaucimobi) prepared on the E. coli JM109 strain obtained in Example 2 using the same medium and culture conditions as in Example 2 was used.
lis) (current name: Sphingomonas pusubimovils)
(FERM P-5122) was used to determine the E. coli JM1
The same concentration was mixed with strain 09 to prepare a composite microorganism system. The obtained mixture (the concentration of the bacterial cells of Escherichia coli JM109 strain was determined in Example 2)
(Same as described above) was prepared in the same manner as in Example 2. Using the nucleic acid probe prepared in Example 3, the excitation light was 543 nm, and the measured fluorescent color was 569 n.
As a result of performing the same experiment as in Example 2 except that
The same results as in Example 2 were obtained.

Example 5 The base selectivity of a target nucleic acid in the fluorescence quenching phenomenon, that is, the base specificity of the present invention was examined. Ten types of target synthetic deoxyribooligonucleotides (30 mer) shown below were prepared using a DNA synthesizer ABI394 (Perkin Elmer, USA).

Further, the following probe of the present invention was prepared in which the deoxyribooligonucleotide corresponding to the above-mentioned synthetic DNA was labeled at the 5 'end with bodepy FL. The primer DNA corresponding to the above-mentioned synthetic DNA, in which-(CH ₂ ) ₆ -NH ₂ is bonded to the phosphate group at the 5 ′ end, was obtained from Medland Certified Reagent Company, USA ) Purchased from. Furthermore, a molecular probe (Molecular Prob
es) from the Fluoro Reporter Kit (FluoReporter K)
its) F-6082 (BODIPY FL propionic acid succini)
dyl esters), and a kit containing a reagent for binding the compound to an amine derivative of an oligonucleotide). The kit was allowed to act on the purchased primer DNA to synthesize probes probe a to d and f to h of the present invention labeled with the following bodypy FL. Then, the extent to which the emission of the fluorescent dye decreased upon hybridization with the corresponding synthetic deoxyribooligonucleotide was examined under the following conditions, and the specificity of the probe of the present invention was examined.

[0098]

[0099]

[0100]

[0101]

(1) Components of Hybridization Solution Synthetic DNA 320 nM (final concentration) Nucleic acid probe 80 nM (final concentration) NaCl 50 mM (final concentration) MgCl ₂ 1 mM (final concentration) Tris-HCl buffer (pH = 7.2) 100 mM (final concentration) Milli-Q pure water 1.6992 ml Final volume 2.000 ml (2) Hybridization temperature: 51 ° C (3) Measurement conditions: Excitation light: 543 nm Measurement fluorescence color: 569 nm

[0103]

[Table 1]

The results are shown in Table 1. As can be seen from Table 1, the nucleic acid probe labeled with a fluorescent dye
When hybridized to A (deoxyribooligonucleotide), G (guanine) is added to the base sequence of the target DNA by 1 to 3 bases away from the base at the end where the probe and target DNA hybridized. This shows that it is preferable that the nucleotide sequence of the probe is designed so that at least one or more nucleotides are present. In addition, when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target DNA, the base sequence of the probe is set so that the base pair of the hybridized product forms at least one pair of G and C at the end. It can be seen from Table 1 that it is preferable that is designed.

Example 6 A target nucleic acid having the following nucleotide sequence and a nucleic acid probe of the present invention were prepared. The influence of the number of G in the target nucleic acid and the number of G in the nucleic acid probe of the present invention was examined in the same manner as in the above example.

[0106]

[0107]

[0108]

[0109]

[0110]

[Table 2]

As can be seen from Table 2, it is recognized that the number of G in the target nucleic acid and the number of G in the probe of the present invention do not significantly affect the decrease in the fluorescence intensity.

Example 7 A target nucleic acid having the following nucleotide sequence and a nucleic acid probe of the present invention were prepared. The effects of the base species in the target nucleic acid and the base species in the nucleic acid probe of the present invention were examined in the same manner as in the above example.

[0113]

[0114]

[0115]

[Table 3]

As can be seen from Table 3 and the above example,
(i) The terminal of the probe of the present invention labeled with a fluorescent dye is C
When the target nucleic acid is hybridized, forms a GC pair, and (ii) is labeled with a fluorescent dye.If the end of the probe of the present invention is composed of a base other than C, it is labeled with a fluorescent dye. From the base pair of the base at the given position and the base of the target nucleic acid, it can be seen that when at least one G is present at the 3 ′ end of the target nucleic acid, the reduction rate of the fluorescence intensity is large.

Example 8 Regarding the type of dye to be labeled on the nucleic acid probe of the present invention,
Investigation was conducted in the same manner as in the above-mentioned Example. The probe of the present invention used the probe z of Example 7 and the target nucleic acid used the oligonucleotide z of Example 7 above. Table 4 shows the results. As can be seen from the table, those suitable as the fluorescent dye used in the present invention are FITC, BODIPY FL,
BODIPY FL / C3, 6-joe, TMR and the like can be mentioned.

[0118]

[Table 4]

Example 9 Preparation of nucleic acid probe: 16SrR of E. coli JM109 strain
(5 ') CAT CCC CAC CTT CC that specifically hybridizes to the 16S rRNA nucleotide sequence of KYM-7 strain corresponding to the nucleotide sequence from the 1156th to 1190th nucleotide of NA
It has a base sequence of T CCC AGT TGACCC CGG CAG TC (3 ′) (35 base pairs), bases 1 to 16 and 25 to 35 are deoxyribonucleotides, and bases 17 to 24 are amino groups at the 2 ′ carbon OH group. A ribooligonucleotide modified with a OH group, wherein the OH group of the phosphate group at the 5 ′ end is modified with — (CH ₂ ) ₇ —NN ₂ and bonded thereto, in the same manner as in Example 1; Medland Certified・ The Resin Company (Midl
and Certified Reagent Company, USA). Further, a molecular probe (Mole
Fluorescent Reporter Kit (Fluo
Reporter Kits) F-6082 (BODIPY FL / C6 propio acid succinimidyl ester of FL / C6)
In addition to nic acid succinimidyl ester), a kit containing a reagent that binds the compound to an amine derivative of an oligonucleotide was purchased. The kit was allowed to act on the oligonucleotide to synthesize a nucleic acid probe labeled with bodypi FL / C6. The obtained synthesized product was purified in the same manner as in Example 1 to obtain a nucleic acid probe labeled with Bodepy FL / C6 in a yield of 23% from the initial oligonucleotide material of 2 mM. This probe was designated as a 35 base chain 2-O-Me probe.

(5 ') AGG CCG GCC CTT GAC TTT CCT
A riboxyoligonucleotide having the base sequence of (3 ′) was synthesized using a DNA synthesizer in the same manner as described above to obtain a forward-type helper probe. On the other hand, (5 ') AUG GGA GUUCAG UAG UAC CCG CAA U
A riboxyoligonucleotide having the base sequence of GC UGG UCC (3 ') was synthesized using a DNA synthesizer in the same manner as described above to obtain a backward type helper probe.

After the above-mentioned 16S rRNA was heated at 95 ° C. for 5 minutes, it was added to a probe solution under the following reaction conditions, and a fluorescence measurement instrument Perkin Elmer (Perkin Elmer) was used.
Elmer) The fluorescence intensity was measured with LS-50B. The result is shown in FIG. In addition, the above-mentioned 16S rRNA without heat treatment
Was used as a control. It can be seen from FIG. 2 that the decrease in the fluorescence intensity is large in the heat-treated experimental plot. This result indicates that the 16S rRNA was heat-treated at 95 ° C. to perform stronger hybridization with the probe of the present invention. Reaction conditions: 16S rRNA: 10.0 nM Probe: 25 nM Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulfite, pH 5.1 Temperature: 70 ° C

Example 10 (Influence of Helper Probe on Hybridization Efficiency) The following probe of the present invention and a helper probe that hybridize to the above 16S rRNA were prepared in the same manner as described above. And, under the following conditions, 2 'of the present invention
The effect of the -O-Me probe, the effect of the base chain length of the probe, and the effect of the helper probe were examined.
The results are shown in A, B, C, and D of FIG. The figure shows that the 2'-O-Me probe of the present invention contributes to the hybridization efficiency. In addition, when the base chain of the 2'-O-Me probe is short, the helper probe is useful for increasing the hybridization efficiency.

1) the same 35 base chain 2'-O-Me probe as described above; 2) the same base sequence as the 35 base chain 2'-O-Me probe as in 1), except that the oligonucleotide is a deoxyribonucleotide. Consisting probe (35 base chain DN
A probe), 3) The same nucleotide sequence as the 35-base chain 2-O-Me probe of 1), but 8 bases from the 5 ′ end and 10 bases from the 3 ′ end.
Probe with nucleotides removed (17 base chain)
4) A probe (17-base DNA probe) having the same base sequence as the 33-base DNA probe of 2) above, but having 16 nucleotides removed from the 3 'end,

5) A helper probe in which the OH group of the central 8 bases of the forward type helper probe is modified with a methyl group (a furd type 2-O-Me helper probe) 6) The central 8 bases of the reverse type helper probe A helper probe in which the OH group is modified with a methyl group (reverse type 2-O-Me helper probe) 7) A helper probe having the same base sequence as that of the forward type helper probe, but the oligonucleotide is composed of deoxynucleotide. Probe (forward type DNA helper probe) 8) Helper probe (reverse type DNA helper probe) having the same nucleotide sequence as that of the reverse type helper probe but composed of deoxyribonucleotides 9) (5 ′) Ribo-oligonucleotide having the nucleotide sequence of GUGACGGUCACUAUUUGACCUCCUUCCACCCC (3 ') (35 bases ribooligonucleotide), 10) (5 ') GUGACGGUCACUAUUUG (3') consisting ribooligonucleotide (17 bases stranded ribonucleic oligonucleotide having a nucleotide sequence),

Reaction conditions: 16S rRNA: 10 nM probe: 25 nM helper probe: 1 μM buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulphite, pH 5.1 Temperature: 35 base chain ribooligonucleotide 2- When using an O-Me probe: 70 ° C. 17 base strand ribooligonucleotide 2-O-Me probe or 33 base strand ribooligonucleotide DNA probe: 65 ° C. 17 base strand ribooligonucleotide DNA probe: 50 ° C.

Example 11 (Preparation of Calibration Curve for RRNA Measurement) After heating the above rRNA in the range of 0.1 to 10 nM at 95 ° C. for 5 minutes, it was added to a reaction solution which was previously under the following reaction conditions.
After 1000 seconds, decrease the fluorescence intensity with the PerkinElmer LS-5.
Measured using 0B. The result is shown in FIG. The figure shows that the calibration curve shows linearity at 0.1 to 10 nM. Reaction conditions: 33 base chain ribooligonucleotide 2-O-Me probe: 1.0-25 nM Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulfite, pH 5.1 Temperature: 70 ° C.

Example 12 (FISH method) Cellulomonas sp.KYM-7 (FERM P-1680)
6) and Agrobacterium sp. KYM-
8 (FERM P-11358) A 35-36 base chain deoxyoligonucleotide 2-O-Me probe of the present invention that hybridizes to each rRNA was prepared in the same manner as described above. The base sequence of each probe is as follows.

[0128] 35 base chain deoxyoligonucleotide 2-O- for measuring rRNA of Cellulomonas sp.KYM-7
Me probe: (5 ') CAT CCC CAC CTT CCT C CG AGT TGA
CCC CGG CAG TC (3 ') (The underlined portion is modified with a methyl group.) Agrobacterium sp.KYM-8
36 base chain deoxyoligonucleotide 2-O-Me probe for rRNA measurement of (FERM P-11358): (5 ') CA
T CCC CAC CTT CCT C TC GGC TTA T CA CCG GCA GTC (3 ')
(The underlined portion is modified with a methyl group.)

[0129] Cellulomonas sp. KYM-7 and Agrobacterium sp. KYM-8 were mixedly cultured in the following medium composition under the following culture conditions, and the culture was collected at each culture time. From them, rRNA was prepared using RNeasy Maxikit (QIAGENE). After heating the rRNA at 95 ° C. for 5 minutes, it was added to a reaction solution previously set under reaction conditions, reacted at 70 ° C. for 1000 seconds, and the fluorescence intensity was measured using a Perkin Elmer LS-50B. The results are shown in FIG. Note that all r
RNA was measured using RiboGreen RNA Kit (company name: molecular probes, location name: Eugene, Oregon, USA). As can be seen, the kinetics of rRNA of each strain was consistent with the kinetics of total rRNA. In addition, the total amount of rRNA of each strain coincided with the total rRNA. This indicates that the method of the present invention is an effective method in the FISH method.

[0130] Medium Composition (g / l): starch, 10.0; aspartic _{acid, 0.1; K 2 HPO 4,} 5.0; KH 2 PO 4, 2.0; MgSO 4 · 7
H ₂ O, 0.2; NaCl, 0.1; (NH ₄ ) ₂ SO ₄ ; 0.1. Medium 100 ml
Was dispensed into a 500 ml conical flask, and the flask was sterilized at 120 ° C. for 10 minutes using an autoclave kettle.

Culture conditions: The above strains were previously cultured on a slant medium. One platinum loop of cells was taken from the slant medium and inoculated into the sterilized conical flask. 30 ° C, 150
The culture was stirred at rpm.

Reaction conditions: 33 base chain deoxyoligonucleotide 2-O-Me probe: 1.0 to 10 nM Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulphite, pH 5.1 Temperature: 70 ° C.

Example 13 below describes a method for analyzing or measuring polymorphisms and mutations in a target nucleic acid or gene. Example 13 Four types of oligonucleotides having the base sequences shown below were synthesized using the DNA synthesizer of Example 5 described above. In the same manner as in Example 5, a nucleic acid probe of the present invention having the following nucleotide sequence was synthesized. After the probe and each oligonucleotide were hybridized in a solution, it was examined whether single base substitution can be evaluated from the change in fluorescence intensity. The nucleotide sequence of the nucleic acid probe of the present invention,
It is designed to match 100% when G is present at the 3 'end of the target oligonucleotide. The hybridization temperature was set at 40 ° C. at which 100% of the base-pairs of the probe and the target oligonucleotide could hybridize. The concentration of the probe and the target oligonucleotide, the concentration of the buffer solution, the fluorescence measurement device, the fluorescence measurement conditions, the experimental operation, and the like are the same as those in Example 5.

[0134]

Table 5 shows the results. From the table, the target oligonucleotide No. In Nos. 1 to 3, no change in the fluorescence intensity was observed, but the target oligonucleotide N
o. In No. 4, an 84% reduction was observed.

[0136]

[Table 5]

In the present invention, a method for analyzing or measuring or measuring or measuring polymorphisms and mutations of target nucleic acids or genes (Nos. 1 to 4) (data in columns A and B in Table 5). The fluorescence intensity value of the reaction system when the target nucleic acid or gene is hybridized with a nucleic acid probe labeled with a fluorescent dye (the above-described nucleic acid probe), Correcting by means the calculation of (AB) / B in Table 3. From the above results, when the target nucleic acid is double-stranded, G → A, G ← A, C → T, C ← T,
It became clear that substitution of G → C and G ← C can be detected.

Embodiment 14 FIG. 6 shows a model of a DNA chip of the present invention. First, the probe of the present invention prepared in Example 13, 3′TTTTTT
TTGGGGGGGGC 5 'BODIPY 3' OH at the 3 'end of FL / C6
An amino group-introduced amino acid is prepared, and a slide glass whose surface is treated with a silane coupling agent having an epoxy group as a reactive group is prepared. The solution containing the probe of the present invention is used as a DNA chip preparing device GM
Spot on the glass slide with S ^TM 417 ARRAYER (TAKARA). Then, the probe of the present invention binds to the glass surface at the 3 'end. The slide glass is placed in a closed container for about 4 hours to complete the reaction. Then, the slide glass is alternately soaked twice in a 0.2% SDS solution and water for about one minute. Further, it is placed in a boron solution (a solution of 1.0 g of NaBH _{4 in} 300 ml of water) for about 5 minutes. After soaking in water at 95 ° C. for 2 minutes, quickly soak the reagent twice in a 0.2% SDS solution and water alternately for about 1 minute to wash away the reagent. Dry at room temperature.
Thus, the DNA chip of the present invention is prepared. Further, by providing a minute temperature sensor and a heater as shown in the figure on the lower surface of the glass of each spot, a high-performance DNA chip of the present invention can be produced. A case where a target nucleic acid or a gene is measured using this DNA chip will be described. When the target nucleic acid or gene is not hybridized to the probe, or when the fluorescent dye-labeled end of the present invention does not form a GC pair even after hybridization, or when the probe is hybridized with the target nucleic acid or gene. If G (guanine) does not exist in the base sequence of the target nucleic acid or gene at least 1 to 3 bases apart from the terminal base portion, the fluorescence intensity does not change. However, upon hybridization, the fluorescence intensity decreases. This fluorescence intensity was measured using a DNA chip analyzer GMS ^™ 41.
It can be measured using an 8 Array Scanner (TAKARA).

The PCR of the present invention is described in Examples 15 to 19 below.
The method is described. Example 15 The 16S rRNA gene in E. coli genomic DNA was used as a target nucleic acid for amplification of the nucleic acid (BODIPY FL /
A primer (labeled with C6) was prepared.

Preparation of primer 1 (Eu800R: reverse type): A deoxyribooligonucleotide having the base sequence of (5 ′) CATCGTTTACGGCGTGGAC (3 ′) was prepared using a DNA synthesizer ABI394.
(Perkin Elmer, USA), and the oligodeoxyribonucleotide was treated with a phosphatase to treat the phosphate group at the 5 'end of the oligodeoxyribonucleotide, to give a cytosine. ₂ ) _9- NH ₂ conjugate was purchased from Midland Certified Raised Company (USA). In addition, from Molecular Probes, a Fluoro Reporter Kit (FluoReporter
Kits) F-6082 (BODIPY FL propionic acid succinimi
kit containing a reagent for binding the compound to the amine derivative of the oligonucleotide in addition to dyl ester). The kit was allowed to act on the purchased oligonucleotide to synthesize primer 1 of the present invention labeled with bodypi FL / C6.

Purification of synthesized product: The obtained synthesized product was dried to obtain a dried product. Add it to a 0.5M Na ₂ CO ₃ / NaHCO ₃ buffer (pH
9.0). The lysate was subjected to gel filtration using a NAP-25 column (manufactured by Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65%, 2
5 minutes) under the following conditions. Then, the eluting main peak was collected. The fractionated fraction was lyophilized to give the primer 1 of the present invention to the first oligonucleotide material 2
Obtained in 50% yield over mM.

The conditions for the above-mentioned 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: CAPCEL PAK _C18 ; 6 × 250 mm Elution rate: 1.0 ml / min Temperature: 40 ° C. Detection: 254 nm

Example 16 Preparation of Primer 2 (Eu500R / forward: forward type): A fluorescent dye (BODIPY FL / C6) was added to the 5 ′ end of a deoxyribooligonucleotide having the base sequence of (5 ′) CCAGCAGCCGCGGTAATAC (3 ′). The labeled primer 2 was used in Example 13
It was prepared in the same manner as in a yield of 50%.

Example 17 Escherichia coli strain JM109 was incubated overnight at 37 ° C. in a test tube containing 5 ml of sterilized nutrient broth (NB) (manufactured by Difco) liquid medium (composition: NB, 0.08 g / 100 ml). The cells were cultured with shaking. 1.5 ml of the culture was centrifuged in a 1.5 ml centrifuge tube to obtain cells. From these cells, DNeasy Tissue
Genomic DNA was extracted using Kit (Qiagen, Germany). The method followed the protocol of this kit.
As a result, a 17 ng / μl DNA solution was obtained.

Example 18 Using the genomic DNA of Escherichia coli, Primer 1 and / or Primer 2, the LightCycler ^™ system (Ligh
PCR reaction was carried out in the usual manner using a tCycler ^™ System). The operation followed the procedure manual of the relevant system equipment. In the above system, PCR is performed according to the present invention instead of the nucleic acid probe (two probes utilizing the FRET phenomenon) and the normal primer (a normal primer not labeled with a fluorescent dye) described in the procedure manual. The procedure was performed according to the procedure except that the primers 1 and / or 2 were used.

PCR was performed with the following components. E. coli genomic DNA solution 3.5 μl (final concentration 0-6 ng / 20 μl) (final copy number 0-2.4.10 ⁶ ) Primer solution 0.8 μl (final concentration 0.08 μM) Taq solution 10.0 μl Milli-Q pure Water 5.7 μl Total volume 20.0 μl Escherichia coli 16S rDNA as the target nucleic acid was used at the concentration of the experimental section shown in the description column of FIG.
Similarly, primers 1 and / or
Or experiments were performed with a combination of the two.

The above Taq solution is a mixture of the following reagents. Taq solution 96.0 μl Milli-Q pure water 68.2 μl Taq DNA polymerase solution 24.0 μl Taq start 3.8 μl

Incidentally, the Taq solution and the Taq DNA polymerase solution are DNS sold by Roche Diagnostics, Inc.
A master hybridization probe (DNA Mast
er Hybridization Probes) kit. Especially Ta
q DNA polymerase solution is 10 × conc. (red cap)
Was used 10 times diluted. Taq Start is a Taq DNA product sold by Clonetech (USA).
It is an antibody for polymerase, and the activity of Taq DNA polymerase can be suppressed up to 70 ° C by adding it to the reaction solution. That is, a hot start can be performed.

The reaction conditions are as follows. Denaturation Initial: 95 ° C., 120 seconds Re: 95 ° C., 0 seconds Annealing conditions 57 ° C., 5 seconds Measurements were performed using a LightCycler ^™ system.
At that time, among the detectors F1 to F3 in the system, F
One detector was used, the gain of the detector was fixed at 10, and the excitation intensity was fixed at 75.

The results are shown in FIGS. 7 and 8, it can be seen that the cycle number at the time when the decrease in the emission of the fluorescent dye is observed is proportional to the copy number of Escherichia coli 16S rDNA of the target nucleic acid. In the figure, the amount of decrease in the emission of the fluorescent dye is expressed as the decrease in the fluorescence intensity.
FIG. 9 shows E. coli 16SrDN as a function of cycle number.
1 is a calibration curve of Escherichia coli 16S rDNA expressing the copy number of A. The correlation coefficient was 0.9973, showing a very good correlation. As can be seen from the above results, the use of the quantitative PCR method of the present invention makes it possible to measure the initial copy number of a target nucleic acid. That is, the target nucleic acid can be measured.

Example 19 In Example 18, PCR was performed using the probe of the present invention as a primer. However, in this example, the probe of the present invention was replaced with the two probes utilizing the FRET phenomenon used in the conventional method. The PCR of the present invention was performed under the following conditions. a) Target nucleic acid: 16S-rDNA of E. coli b) Primers used: • Forward primer E8F: (3 ') AGAGTTTGATCCTGGCTC
AG (5 ') ・ Reverse primer E1492R: GGTTACCTTGTTACGACTT
(5 ') c) Probe used: BODIPY FL- (3') CCTTCCCACATCGTTT
(5 ') d) PCR measuring instrument used: LightCycler ^™ system e) PCR conditions: Denaturation reaction: 95 ° C for 0 seconds (60 seconds, 95 ° C) Annealing reaction: 50 ° C for 5 seconds Nucleic acid extension reaction: 72 ° C for 70 seconds Total number of cycles: 70 cycles

F) Fluorescence measurement (measured once after each cycle after annealing reaction and denaturation reaction) g) Composition of reaction solution: total amount of reaction solution: 20 μl amount of DNA polymerase (TaKaRa Ex taq): 0.5 U Taq start (antibody): 0.3 μl Primer concentration: 0.2 μM (both) Probe concentration: 0.05 μM MgCl ₂ concentration: 2 mM BSA (bovine serum albumin) concentration: 0.25 mg / ml dNTPs concentration: 2.5 mM (each nucleotide) about).

FIG. 10 shows the result. From the figure, it can be seen that the cycle number at which the decrease in the emission of the fluorescent dye is observed is proportional to the copy number of Escherichia coli 16S rDNA of the target nucleic acid. As can be seen from the above results, the use of the quantitative PCR method of the present invention makes it possible to measure the initial copy number of a target nucleic acid. That is, the target nucleic acid can be measured.

Next, the data analysis method of the present invention for analyzing data obtained by using the above-described quantitative PCR method of the present invention is described in the following examples. Example 20 Human genomic DNA (human β-globin (TaKa
Ra catalog product number 9060) (TaKaRa Corporation)
(Hereinafter, referred to as human genomic DNA) as a target nucleic acid, a primer labeled with Bodepy FL / C6 for amplification of the nucleic acid was prepared.

Preparation of primer KM38 + C (reverse type):
(5 ′) A deoxyribooligonucleotide having a base sequence of CTGGTCTCCTTAAACCTGTCTTG (3 ′) was converted to a DNA synthesizer ABI394.
(Perkin Elmer, USA), and the oligodeoxyribonucleotide was treated with a phosphatase at the 5′-terminal phosphate group to produce cytosine. The 5′-carbon OH group of cytosine was replaced with-(CH ₂ ) _9- NH ₂ was purchased from Midland Certified Residential Company. Furthermore, from Molecular Probes Inc., a reporter kit (FluoReporter Kits) F-6082 (Succinimidyl propionate of BODEPI FL / C6 (BO
DIPY FL propionic acid succinimidylesters)
A kit containing a reagent for binding the compound to an amine derivative of an oligonucleotide). The kit was allowed to act on the purchased oligonucleotide to synthesize a primer KM38 + C labeled with the bodypy FL / C6 of the present invention.

Purification of the synthesized product: The obtained synthesized product was dried to obtain a dried product. 0.5M Na ₂ CO ₃ / NaH ₂ CO ₃ buffer (pH 9.0)
Was dissolved. The lysate was subjected to gel filtration using a NAP-25 column (Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65% for 25 minutes) was performed under the following conditions. Then, the eluting main peak was collected. The fractionated fraction is freeze-dried and the primer of the present invention KM38 + C
Was obtained in 50% yield from the initial oligonucleotide starting material 2 mM.

The conditions for the above-mentioned 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: CAPCEL PAK C18; 6 × 250mm Elution rate: 1.0ml / min Temperature: 40 ° C Detection: 254nm

Example 21 Preparation of primer KM29 (forward type): (5 ′) GGTTGGCC
A deoxyribooligonucleotide having the nucleotide sequence of AATCTACTCCCAGG (3 ′) was synthesized in the same manner as in Example 18.

Comparative Experimental Example 1 (Experimental example using data analysis software having no arithmetic processing step of the present invention for dividing the fluorescence intensity value during the nucleic acid extension reaction using the fluorescence intensity value during the thermal denaturation reaction) ). The above human genomic DNA, primer
Using KM38 + C and primer KM29, a PCR reaction was performed using a LightCycler ^™ system, and the fluorescence intensity in each cycle was measured. The PCR of this comparative example
Is a novel real-time quantitative PCR method that uses a primer labeled with a fluorescent dye described above and measures the decrease instead of the increase in fluorescence emission. Data analysis software was used for the system. In the above system, PCR is performed using the primer KM38 of the present invention in place of the nucleic acid probe (two probes utilizing the FRET phenomenon) and a normal primer (a normal primer not labeled with a fluorescent dye) described in the procedure manual. Except for using + C and KM29, the procedure was performed according to the procedure manual of the apparatus.

The PCR was performed with the following components. Human genomic DNA 1.0 μl (final concentration: 1 to 10,000 copies) Primer solution 4.0 μl (final concentration 0.1 μM) Taq solution 10.0 μl Milli-Q pure water 5.0 μl Total volume 20.0 μl The human genomic DNA is shown in the description column of FIG. The experiment was performed at the concentration of the experimental section. The final concentration of MgCl ₂ was 2 mM.

The Taq solution is a mixed solution of reagents. Taq solution 96.0μl Milli-Q pure water 68.2μl Taq DNA polymerase 24.0μl Taq start 3.8μl

The Taq solution and the Taq DNA polymerase solution were obtained from Roche Diagnostics, Inc.
A master hybridization probe (DNA Mast
er Hybridization Probes) kit. Particularly, the Taq DNA polymerase solution was used by diluting 10 × conc. (Red cap) 10-fold. In addition, Taq Start is a Taq product sold by Clonetech (USA).
This is an antibody for q DNA polymerase. By adding it to the reaction solution, the activity of Taq DNA polymerase can be suppressed up to 70 ° C. That is, a hot start can be performed.

The reaction conditions are as follows. Initial denaturation reaction: 95 ° C., 60 seconds Redenaturation reaction: 95 ° C., 10 seconds Annealing reaction: 60 ° C., 5 seconds DNA extension reaction: 72 ° C., 17 seconds The measurement was performed using a LightCycler ^™ system.
At that time, among the detectors F1 to F3 in the system, F
One detector was used, the gain of the detector was fixed at 10, and the excitation intensity was fixed at 75.

FIG. 11 shows the results of actual measurement of the fluorescence intensity of each cycle by performing PCR as described above. That is, the fluorescence intensity of each copy number of the human genomic DNA at the time of the denaturation reaction and the nucleic acid extension reaction of each cycle was measured and printed. In each cycle, the fluorescence intensity value is constant during the denaturation reaction, but 2 times during the nucleic acid extension reaction.
It is observed that the fluorescence intensity decreases from around the fifth cycle. Thus, it can be seen that the decrease occurs in the order of the copy number of the human genomic DNA.

In FIG. 11, the fluorescence intensity value at the initial cycle number is not uniform for each copy number of the human genomic DNA. Therefore, the following process was added to the data analysis method used in this experimental example. (B) The process of converting the fluorescence intensity value of each cycle with the fluorescence intensity value of the 10th cycle as 1, that is, the process of calculating by the following [Equation 8], C _n = F _n (72) / F ₁₀ ( 72) [Expression 8] where C _n = converted value of each cycle value, F _n (72) = fluorescence intensity value of each cycle at 72 ° C., F ₁₀ (72) = fluorescence intensity value of the 10th cycle at 72 ° C. . (C) plotting the converted value obtained in the step (b) with respect to each cycle number, and displaying and / or printing on a display;

(D) a step of calculating a change rate (decrease rate, extinction rate) of the fluorescence intensity from the converted value of each cycle obtained in the step (b) according to the following [Equation 9], [Equation 9] F _dn = log _{10 −100} −C _n × 100)｝ F _dn = 2log ₁₀ ｛1−C _n ｝ where, F _dn = fluorescence intensity change rate (decrease rate, extinction rate), C
_n = value obtained by [Equation 8]. (E) plotting the converted value obtained in the step (d) with respect to each cycle number, and displaying and / or printing on a display;

(F) Of the data processed in step (d), 0.5 is set to a threshold (thresh).
hold) and calculating the number of cycles that have reached the value. (g) Create a graph in which the value calculated in step (f) is plotted on the X-axis and the copy number before the start of the reaction is plotted on the Y-axis. (H) displaying and / or printing the graph created in the step (g) on the display; and (j) calculating the correlation coefficient or relational expression of the straight line drawn in the step (h). (I) displaying and / or printing the calculated value or the relational expression calculated in the step (j) on the display.

Using the data analysis software described above, the data obtained in FIG. 11 was processed as follows following the above. FIG. 12 is a printout of the data processed in the process (b) (the process (c)). That is, the fluorescence intensity value at the 10th cycle is converted as 1, and the converted value is plotted against the number of cycles. FIG. 13 shows the data processed in the step (d) printed (the step (e)). That is, the reduction rate (quenching rate) of the fluorescence intensity was calculated from each plot value in FIG. 12, and each calculated value was plotted against each cycle number.

FIG. 14 is a graph obtained by printing the graph created in the step (g) on the data processed in the step (f) (the step (h)). That is, the threshold of the fluorescence intensity reduction rate = 0.5
d) A graph in which the number of cycles reaching the value is plotted on the X axis, and the copy number of the human genomic DNA before the start of the reaction is plotted on the Y axis. The correlation coefficient (R2) of the straight line in this graph was calculated in the process of (j) and printed (process of (i)), and was 0.9514. Thus, it was impossible to obtain an accurate copy number with this correlation coefficient.

Example 22 (Experimental example in which data processing was performed using the data analysis method of the present invention) PCR was performed in the same manner as in Comparative experimental example 1. The data processing was performed in the same manner as in Comparative Experimental Example 1 except that the following process (a) was performed before the process of (b) in Comparative Experimental Example 1, and the processes in (b) and (d) were changed as follows. A similar process was performed. (A) The fluorescence intensity value of the reaction system when the amplified nucleic acid in each cycle was hybridized with a nucleic acid primer labeled with a fluorescent dye (that is, the fluorescence intensity value at the time of nucleic acid extension reaction (72 ° C.)) was amplified. Fluorescence intensity value of the reaction system when the nucleic acid hybridized with the nucleic acid primer is dissociated (ie, the fluorescence intensity value at the time of the nucleic acid thermal denaturation reaction (95 ° C.))
, Ie, the measured fluorescence intensity value was corrected by [Equation 1]. f _n = f _{hyb, n} / f _{den, n} [Equation 1] [where, f _n = correction value of the fluorescence intensity of the cycle, f _{hyb, n} =
The fluorescence intensity value at 72 ° C. in each cycle, f _{den, n} = the fluorescence intensity value at 95 ° C. in each cycle] FIG. 15 shows the obtained values plotted for each cycle number.

(B) An arithmetic processing step of substituting the correction operation processing value in [Equation 1] in each cycle into [Equation 3] and calculating a fluorescence change rate (decrease rate or extinction rate) between samples in each cycle. That is, the following [Equation 1]
0], F _n = f _n / f ₂₅ [Equation 10] [where, F _n = operation processing value of each cycle, f _n = value of each cycle obtained by [Equation 1], f ₂₅ = value obtained by [Equation 1] and the number of cycles is 25]. [Equation 1
0] is obtained when a = 25 in [Equation 3].

(D) calculating the logarithmic value of the change rate (decrease rate or extinction rate) of the fluorescence intensity according to [Equation 6] with the calculated value of each cycle obtained in the step (b);
That is, a process of performing the arithmetic processing by the following [Equation 11], log ₁₀ {(1-F _n ) × 100} [Equation 11] [where, F _n = value obtained by [Equation 10]]. [Equation 1
1] is a case where b = 10 and A = 100 in [Equation 6].

The above results are shown in FIGS. FIG. 16 is a graph in which the values processed in the processes (a) and (b) are plotted against the number of cycles and printed.
FIG. 17 plots the values calculated in step (d) for the values obtained in FIG. 16 against the number of cycles,
It is printed.

Next, based on the graph of FIG. 17, processing was performed in the processes (f), (g), and (h). That is,
Similarly to Comparative Example 1 based on the graph of FIG. 17, log ₁₀
(Fluorescent intensity change rate)
0.1, 0.3, 0.5, 0.7, 0.9 and 1.2 were selected, and the number of cycles that reached that value was plotted on the X-axis and the human genome D
The copy number before the start of the NA reaction was plotted on the Y-axis, and a calibration curve was drawn. FIG. 18 shows the result. Correlation coefficients (R ² ) obtained by processing these calibration curves in the processes (j) and (i) are 0.998, 0.999, 0. 999
3, 0.9985, 0.9989 and 0.9988. From these correlation coefficients, it was recognized that it is desirable to adopt 0.5 (correlation coefficient 0.9993) as the threshold value. With the calibration curve having this correlation coefficient, it can be seen that the copy number before the start of the reaction can be accurately determined for the nucleic acid sample having the unknown copy number.

Example 23 (Melting curve analysis of nucleic acid and Tm
Example of Value Analysis) For the nucleic acid amplified by the novel PCR method of the present invention, 51) a process of gradually increasing or decreasing the temperature from a low temperature until the nucleic acid is completely denatured (for example, 50 ° C.)
To 95 ° C.), 52) a step of measuring the fluorescence intensity at short time intervals (for example, 0.2 ° C. to 0.5 ° C.) in the step 51), and 53) a measurement result of the step 52). Displaying on the display every time, that is, displaying the melting curve of the nucleic acid; 54) firstly differentiating the melting curve of the above 53) step; 55) differential value (-dF / dT, F: Fluorescence intensity, T: Time) on the display; 56) Software for analyzing the data of the present invention, comprising the step of obtaining an inflection point from the differential value obtained in 55). Combined with wear. A novel real-time quantitative PCR reaction of the present invention was performed using the LightCycler ^™ system in which a computer-readable recording medium on which the data analysis software was recorded was installed, and a nucleic acid melting curve was analyzed. In the present invention, the fluorescence intensity increases as the temperature increases.

The same PCR as in Example 20 was performed on 1 and 10 copies of the same human genomic DNA as in Example 22.
And 51), 52), 53), 54) and 5
FIG. 1 shows a printout of the data processed in step 5).
9 FIG. 20 is a diagram of a nucleic acid melting curve obtained by treating the 75th amplification product of 1 copy and 10 copies in the steps 51), 52) and 53) of this example. FIG. 21 shows the result of differentiating this curve in the process of 54) and obtaining the inflection point (Tm value) in the processes of 55) and 56). From FIG. 21, it was found that the amplification products of one copy and ten copies had different Tm values, so that each amplification product was a different product.

[0177]

The present invention has the following effects. 1) As described above, the use of the nucleic acid measurement method of the present invention, the probe and the device of the present invention do not require any operation such as removing unreacted nucleic acid probes from the measurement system. It can be measured easily. Further, when applied to a complex microbial system or a symbiotic microbial system, the abundance of a specific strain in the system can be measured specifically and in a short time. Also,
The present invention provides a simple method for analyzing or measuring polymorphisms or mutations such as SNPs of a target nucleic acid or gene. 2) The quantitative PCR method of the present invention has the following effects. a. Since no factor that inhibits the amplification of the target nucleic acid by Taq DNA polymerase is added, quantitative PCR can be performed under the same conditions as those of conventionally known ordinary PCR having specificity. b. In addition, since the specificity of the PCR can be kept high, the amplification of the primer dimer is slowed, so that the quantification limit is reduced by about one order as compared with the conventionally known quantitative PCR. c. Since there is no need to prepare a complicated nucleic acid probe, the time and cost required for the preparation can be saved. d. The effect of amplifying the target nucleic acid is large, and the amplification process can be monitored in real time. 3) When analyzing data obtained by the real-time quantitative PCR method, a calibration line for obtaining the copy number of a nucleic acid for a nucleic acid sample having an unknown nucleic acid copy number is prepared using the data analysis method of the present invention. The correlation coefficient of the line is much higher than that obtained by the conventional method. Thus, using the data analysis method of the present invention, the exact copy number of a nucleic acid can be determined. 4) Data analysis software according to the method for analyzing data obtained by the real-time quantitative PCR method of the present invention, and a computer-readable recording medium recording the procedure of the analysis method as a program;
In addition, when a real-time quantitative PCR measurement or analysis device using the same is used, a calibration line having a high correlation coefficient can be automatically created. 5) In addition, by using the novel nucleic acid melting curve analysis method of the present invention, a highly accurate Tm value of a nucleic acid can be obtained. Furthermore, using the software for data analysis according to the method, a computer-readable recording medium in which the procedure of the analysis method is recorded as a program, and a real-time quantitative PCR measurement or analysis device using the same, Accurate Tm value can be obtained

[Brief description of the drawings]

FIG. 1 shows that the nucleic acid probe obtained in Example 1 is used to count 335 to 358 from the 5 ′ end of 16S rRNA of E. coli.
The figure which shows the fluorescence intensity measurement data at the time of measuring the 3rd nucleic acid base sequence.

FIG. 2: Effect of heat treatment on hybridization of 35 base strand 2-o-Me probe to target nucleic acid Dotted line: After rRNA was heat-treated, target nucleic acid was added. Solid line: unheated rRNA.

FIG. 3 shows the effect of the number of bases in the base chain of the probe, the helper probe, and the modification of the methyl group at the 2′-OH group of 2′-ribose at the 5 ′ end of the probe on the hybridization between the probe and 16S rRNA of the target nucleic acid.

FIG. 4: Calibration curve for measuring rRNA by the method of the present invention

FIG. 5: rR of a combined culture system of KYM7 strain and KYM8 strain
Analysis of time course of NA amount by FISH method of the present invention

FIG. 6 is a diagram showing a DNA chip of the present invention.

FIG. 7 is a diagram showing a quantitative PCR method using primers 1 and 2 labeled with Bodepy FL: the relationship between the number of cycles and the decrease in the emission of the fluorescent dye. The symbol “-” in the figure represents the following meaning.

FIG. 8 is a graph showing the relationship between the number of cycles and the logarithmic value of the amount of decrease in the emission of a fluorescent dye, in a quantitative PCR method using primers 1 and 2 labeled with bodypy FL. The symbol "-" in the figure represents the same meaning as in FIG.

FIG. 9 shows a calibration curve of Escherichia coli 16S rDNA prepared using the quantitative PCR method of the present invention. n: 10 ⁿ

FIG. 10 shows the case where real-time quantitative PCR was performed using one 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. It is a figure which shows the change of the phenomenon rate of fluorescence intensity. The figure below shows the number of cycles (thresholdnumbe) at which the phenomenon of fluorescence intensity begins to be significantly observed.
(r: Ct value) was calculated and a calibration curve was created.

FIG. 11 is a diagram showing a fluorescence reduction curve obtained by real-time quantitative PCR using a primer labeled with the bodypy FL of the present invention without performing the correction arithmetic processing of the present invention. ■: target nucleic acid = 10 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C. ●: target nucleic acid = 100 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C. ▲: target nucleic acid = 1000 copies; Reaction system temperature = 72 ° C. ◆: target nucleic acid = 10000 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C. □: target nucleic acid = 10 copies; temperature of reaction system for fluorescence intensity measurement = 95 ° C. ○: target nucleic acid = 100 copies; temperature of reaction system for measuring fluorescence intensity = 95 ° C. Δ: target nucleic acid = 1000 copies; temperature of reaction system for measuring fluorescence intensity = 95 ° C. Δ: target nucleic acid = 10000 copies; temperature of reaction system for measuring fluorescence intensity = 95 ° C

12 is a diagram showing a fluorescence reduction curve of real-time quantitative PCR obtained in the same manner as in the case of the curve in FIG. 11, except that the value at the 10th cycle of each curve is corrected to 1. ■: Target nucleic acid = 10 copies; fluorescence intensity measurement temperature = 72 ° C. ●: target nucleic acid = 100 copies; fluorescence intensity measurement temperature = 72
° C: Target nucleic acid = 1000 copies; Fluorescence intensity measurement temperature = 7
2 ° C ◆: target nucleic acid = 10000 copies; fluorescence intensity measurement temperature =
72 ° C. 5: Target nucleic acid = 10000 copies; fluorescence intensity measurement temperature =
72 ° C

FIG. 13 shows plot values of each curve in FIG.
The figure which shows the curve which calculated the fluorescence intensity reduction rate (change rate) of [Formula 9], and plotted the calculated value. ■: Target nucleic acid = 10 copies ●: Target nucleic acid = 100 copies ▲: Target nucleic acid = 1000 copies ◆: Target nucleic acid = 10,000 copies

FIG. 14 shows a human genome DN obtained from the data shown in FIG.
The figure which shows the calibration straight line of A. y = human β-globin gene copy number x = cycle number (Ct) R ² = correlation coefficient

15 is a diagram showing a curve obtained by plotting a value obtained by performing a correction operation process on the measured value of each cycle in FIG. 11 using [Equation 1] with respect to each cycle number. ■: Target nucleic acid = 10 copies ●: Target nucleic acid = 100 copies ▲: Target nucleic acid = 1000 copies ◆: Target nucleic acid = 10,000 copies

FIG. 16 is a view showing a curve obtained by plotting a value obtained by performing an arithmetic processing on the arithmetic processing value of each cycle in FIG. ■: Target nucleic acid = 10 copies ●: Target nucleic acid = 100 copies ▲: Target nucleic acid = 1000 copies ◆: Target nucleic acid = 10,000 copies

FIG. 17 is a diagram showing a curve obtained by plotting a value obtained by performing an arithmetic processing value of each cycle in FIG. 16 by the equation of [Equation 6] with respect to the number of cycles; ■: Target nucleic acid = 10 copies ●: Target nucleic acid = 100 copies ▲: Target nucleic acid = 1000 copies ◆: Target nucleic acid = 10,000 copies

FIG. 18 shows Ct values as candidates of 0.1, 0.3, 0.5, 0.7, 0.
9 is a diagram in a case where 9 and 1.2 are appropriately selected and a calibration straight line corresponding thereto is drawn. In addition, the correlation coefficient in each calibration curve was shown below. ：: log ₁₀ (fluorescence change rate) = 0.1; correlation coefficient = 0.9
98 1: log ₁₀ (fluorescence change rate) = 0.3; correlation coefficient = 0.9
99 ●: log ₁₀ (fluorescence change rate) = 0.5; correlation coefficient = 0.9
993 Δ: log ₁₀ (fluorescence change rate) = 0.7; correlation coefficient = 0.9
985 □: log ₁₀ (fluorescence change rate) = 0.9; correlation coefficient = 0.9
989 ○: log ₁₀ (fluorescence change rate) = 1.2; correlation coefficient = 0.9
988

FIG. 19: One copy and 10 copies of human genomic DNA
The figure which shows the fluorescence reduction curve at the time of performing real-time quantitative PCR using the primer labeled with the bodypy FL of this invention about (3). However, the correction calculation processing of [Equation 1] was performed. 1: Target nucleic acid = 0 copy 2: Target nucleic acid = 1 copy 3: Target nucleic acid = 10 copies

FIG. 20 is a view showing a melting curve of a nucleic acid when a melting curve analysis of the nucleic acid is performed on the PCR amplification product shown in FIG. 19; 1: Target nucleic acid = 0 copy 2: Target nucleic acid = 1 copy 3: Target nucleic acid = 10 copies

FIG. 21 is a view showing a curve indicating a Tm value obtained by differentiating the curve of FIG. 20 (a valley is a Tm value). 2: Target nucleic acid = 1 copy 3: Target nucleic acid = 10 copies

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/542 G01N 33/566 33/566 C12N 15/00 ZNAA (31) Priority claim number Japanese Patent Application No. 2000- 28896 (P2000-28896) (32) Priority date February 1, 2000 (2000.2.1) (33) Countries claiming priority Japan (JP) (reported by applicant) (72) Kurane, the inventor of the Special Energy Act, the New Energy and Industrial Technology Development Mechanism, the Development of Biological Resource Utilization Technology such as Complex Biological Systems, and the Revitalization of Industrial Vitality. Ryuichiro 1-3-1 Higashi, Tsukuba, Ibaraki Pref. Industrial Technology Research Institute of Biotechnology and Industrial Technology (72) Inventor Takahiro Kanakawa 1-3-1 Higashi, Tsukuba, Ibaraki Pref. Within the Technology Research Institute (72) Inventor Yoichi Kamagata 1-3-1 Higashi, Tsukuba, Ibaraki Pref. Within the Research Institute of Biotechnology and Industrial Technology (72) Inventor Shinya Kurata 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environmental Engineering (72) Inventor Kazutaka Yamada 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environmental Engineering Co., Ltd. (72) Toyoichi Yokomaku 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environment Engineering Co., Ltd. (72) Inventor Osamu Oyama 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environmental Engineering Co., Ltd. (72) Kenta Furusho 1-9-8, Higashikanda, Chiyoda-ku, Tokyo Environment Engineering Co., Ltd. F-term (reference) 2G042 AA01 BD12 BD20 CB03 DA08 FA11 FB05 FC01 4B024 AA11 AA19 CA01 CA09 CA11 HA13 4B029 AA07 AA23 FA10 FA12 FA15 4B063 QA01 QA12 QQ06 QQ08 QQ52 QQ54 QR32 QR32 QR41 QR08 QR32

Claims (45)

[Claims] In a nucleic acid measuring method using a nucleic acid probe labeled with a fluorescent dye, when the nucleic acid probe hybridizes to a target nucleic acid, the fluorescent dye is:
What is claimed is: 1. A nucleic acid probe for reducing its luminescence, comprising: hybridizing the nucleic acid probe to a target nucleic acid; and measuring a decrease in luminescence of a fluorescent dye before and after hybridization. 2. When a nucleic acid probe labeled with a fluorescent dye hybridizes to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces the emission thereof, and the probe is a fluorescent dye at the end thereof. When the nucleic acid probe hybridizes to the target nucleic acid, the terminal sequence is separated from the terminal base by which the probe and the target nucleic acid have hybridized by 1 to 3 bases at the terminal portion, and the base sequence of the target nucleic acid is G A nucleic acid probe for nucleic acid measurement, wherein the nucleotide sequence of the probe is designed so that (guanine) has at least one base. 3. The nucleic acid probe for nucleic acid measurement according to claim 2, wherein the nucleic acid probe is labeled with a fluorescent dye at the 3 ′ end. 4. The nucleic acid probe for nucleic acid measurement according to claim 2, wherein the nucleic acid probe is labeled at a 5 ′ end with a fluorescent dye. 5. The method according to claim 5, wherein the fluorescent dye is a nucleic acid probe that reduces its emission when the nucleic acid probe labeled with the fluorescent dye is hybridized to a target nucleic acid, and the probe has a fluorescent dye at its terminal. When the nucleic acid probe hybridizes to the target nucleic acid, the base pair of the hybridization forms at least one pair of G (guanine) and C (cytosine) at the terminal portion,
A nucleic acid probe for nucleic acid measurement, wherein the nucleotide sequence of the probe is designed. 6. The nucleic acid probe, wherein the 3 ′ terminal base is G or C.
And the 3 ′ end is labeled with a fluorescent dye.
The nucleic acid probe for nucleic acid measurement according to item 1. 7. The nucleic acid probe according to claim 5, wherein the 5 ′ terminal base is G or C.
And the 5 ′ end is labeled with a fluorescent dye.
The nucleic acid probe for nucleic acid measurement according to item 1. 8. The nucleic acid probe according to claim 4, wherein a 3′-carbon hydroxyl group of 3′-terminal ribose or deoxyribose or a 3′-carbon hydroxyl group of 3′-terminal ribose is phosphorylated. Nucleic acid probe for nucleic acid measurement. 9. The oligoribonucleotide of the nucleic acid probe for nucleic acid measurement is 2-o-methyl oligoribonucleotide.
(2-o-methyloligoribonucleotide), PNA, or
The nucleic acid probe for nucleic acid measurement according to any one of claims 1 to 8, which is another chemically modified nucleic acid. 10. The oligoribonucleotide of a nucleic acid probe for nucleic acid measurement, wherein the oligoribonucleotide contains ribonucleotides and deoxyribonucleotides.
The nucleic acid probe for nucleic acid measurement according to any one of claims 2 to 8, which is an ic oligonucleotide). 11. The nucleic acid probe according to claim 10, wherein the ribonucleotide is 2-o-methyl oligoribonucleotide. 12. A nucleic acid probe for nucleic acid measurement according to any one of claims 2 to 8, which is hybridized to a target nucleic acid, and a decrease in the emission of a fluorescent dye before and after hybridization is measured. Method for measuring nucleic acid to be used. 13. The nucleic acid probe for nucleic acid measurement according to claim 9, wherein the nucleic acid probe is hybridized to a target nucleic acid, and the amount of decrease in emission of a fluorescent dye before and after hybridization is measured. Method for measuring nucleic acid. 14. A target nucleic acid or gene polymorphism (polymo
In a method for analyzing or measuring rphism or / and mutation, the nucleic acid probe according to any one of claims 2 to 11 is hybridized to a target nucleic acid or gene, and the amount of change in fluorescence intensity is measured. A method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid or gene, characterized in that: 15. A polymorphism or / and / or a target nucleic acid or gene.
And a measurement kit for analyzing or measuring a mutation, wherein the nucleic acid probe according to any one of claims 2 to 11 is analyzed and the polymorphism and / or mutation of a target nucleic acid or gene is analyzed or measured. kit. 16. A method for analyzing data obtained by the method according to claim 14, wherein the fluorescence intensity value of the reaction system when the target nucleic acid or gene hybridizes with a nucleic acid probe labeled with a fluorescent dye, 3. A data analysis method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid or gene, the method comprising a step of performing a correction process based on the fluorescence intensity value of the reaction system when not hybridized. 17. A polymorphism or / and / or a target nucleic acid or gene.
And in a measuring device for analyzing or measuring mutations,
A measurement device for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid or gene, comprising a means for performing the data analysis method according to claim 16. 18. A computer-readable recording medium on which a program for causing a computer to execute the correction process according to claim 16 is recorded. 19. A helper probe is added to a hybridization reaction system for performing a hybridization reaction before the hybridization reaction.
3. The method for measuring nucleic acid according to item 1. 20. The nucleic acid measuring method according to claim 13, wherein the nucleic acid probe is hybridized with the target nucleic acid after the target nucleic acid is subjected to a heat treatment under conditions suitable for sufficiently destroying its higher-order structure. 21. The kit according to claim 15, wherein a helper probe is added to the hybridization reaction system. 22. The method according to claim 12, wherein the target nucleic acid is RNA.
21. The nucleic acid measurement method according to 13, 19, or 20. 23. The nucleic acid probe according to any one of claims 2 to 11, or two different fluorescent dyes in one molecule, and when not hybridized to a target nucleic acid, Although quenched or emitted by the interaction of the fluorescent dye, a nucleic acid probe having a structure designed to emit or quench when hybridized to the target nucleic acid is bound to the surface of the solid support, and the target nucleic acid is hybridized thereto. A device for measuring a nucleic acid, wherein the device can measure a target nucleic acid. 24. The device for nucleic acid measurement (chip) according to claim 23, wherein the nucleic acid probes are arranged and bound in an array on the surface of the solid support. 25. For each nucleic acid probe bound to the surface of the solid support, at least one temperature sensor and heater are provided on the opposite surface, and the temperature is adjusted so that the nucleic acid probe binding region has an optimal temperature condition. Claim 2 to obtain
25. The device for measuring or detecting a nucleic acid according to 3 or 24. 26. The nucleic acid probe according to claim 23, wherein the nucleic acid probe is bound to the surface of the solid support at an end not labeled with a fluorescent dye.
6. The device for nucleic acid measurement according to any one of 5. 27. A method for measuring a nucleic acid, comprising measuring a target nucleic acid using the nucleic acid measuring device according to claim 23. 28. The method of claim 1, 12, 13, 19 to 22,
28. The method for measuring a nucleic acid according to any one of claims 27, wherein the target nucleic acid is derived from a microorganism or an animal obtained by pure separation. 29. The nucleic acid according to any one of claims 1, 12, 13, 19 to 22, and 27, wherein the target nucleic acid is a nucleic acid of intracellular or cell homogenate of a complex microbial system or a symbiotic microbial system. Measurement method. 30. The PCR method according to claim 2, wherein
A reaction is carried out using the nucleic acid probe according to any one of 5, 6, 8 to 11, and the reaction system in which the probe is decomposed and removed by a polymerase during a nucleic acid extension reaction or during a nucleic acid denaturation reaction or during a nucleic acid denaturation reaction Measure the fluorescence intensity value of the completed reaction system and the fluorescence intensity value of the reaction system when the target nucleic acid or amplified target nucleic acid is hybridized with the nucleic acid probe, and calculate the decrease rate of the fluorescence intensity value from the former A method for measuring a nucleic acid amplified by PCR. 31. The PCR method according to claim 4 or 7,
Perform a reaction using the nucleic acid probe described in the primer,
Measure the fluorescence intensity value of the reaction system when the probe and the target nucleic acid or the amplification target nucleic acid are not hybridized and the reaction system when the nucleic acid probe is hybridized with the target nucleic acid or the amplification target nucleic acid, A method for measuring a nucleic acid amplified by PCR, comprising calculating the former reduction rate of the fluorescence intensity value. 32. The PCR method is a real-time quantitative PC.
The method for measuring an amplified nucleic acid according to the PCR method according to claim 30 or 31, which is an R method. 33. Claims 1, 12, 13, 19 to 22, 2.
In a method for analyzing data obtained by the nucleic acid measurement method according to any one of 7 to 32, the fluorescence intensity value of the reaction system when the target nucleic acid is hybridized with a nucleic acid probe labeled with a fluorescent dye, A data analysis method for a nucleic acid measurement method, wherein the correction is performed based on a fluorescence intensity value of a reaction system when the hybridized product is dissociated. 34. The method for analyzing data obtained by the real-time quantitative PCR method according to claim 32, wherein the amplified nucleic acid in each cycle binds to a fluorescent dye, or the amplified nucleic acid is labeled with a fluorescent dye. The fluorescence intensity value of the reaction system when hybridized with the nucleic acid probe,
Alternatively, data for a real-time quantitative PCR method, comprising an arithmetic processing step of correcting the fluorescence intensity value of the reaction system when the hybridized product is dissociated (hereinafter referred to as a correction arithmetic processing step). analysis method. 35. The data analysis method for a real-time quantitative PCR method according to claim 34, wherein the correction operation processing step according to claim 34 is based on the following [Equation 1] or [Equation 2]. 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 when the amplified nucleic acid binds to the fluorescent dye or when the amplified nucleic acid hybridizes to the nucleic acid probe labeled with the fluorescent dye in n cycles The fluorescence intensity value of the system, f _{den, n} : the combination in n cycles,
Alternatively, the fluorescence intensity of the reaction system when the hybridized product is dissociated]. 36. The method for analyzing data obtained by the real-time quantitative PCR method according to claim 32, wherein the expression (1) or (2) in each cycle.
Real-time quantification characterized by substituting the correction calculation processing value in the above into the following [Equation 3] or [Equation 4], calculating the fluorescence change rate or the fluorescence change rate between each sample in each cycle, and comparing them. Analysis method for dynamic PCR method. F _n = f _n / f _a [Equation 3] F _n = f _a / f _n [Equation 4] [where, F _n : change in fluorescence calculated by [Equation 3] or [Equation 4] in n cycles Ratio or change rate of fluorescence, f _n : Correction processing value by [Equation 1] or [Equation 2] f _a : Change of f _n by correction processing value by [Equation 1] or [Equation 2] Any number of cycles before). 37. The method for analyzing data obtained by the real-time quantitative PCR method according to claim 32, wherein: 1) the change ratio of fluorescence or the change ratio of fluorescence calculated by [Equation 3] or [Equation 4]; Using the data,
The process of performing arithmetic processing according to [Equation 5], [Equation 6] or [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] [where A, b: Arbitrary numerical value, F _n : Fluorescence change rate or fluorescence change rate in n cycles calculated by [Equation 3] or [Equation 4], 2) The number of cycles in which the operation processing value of 1) has reached a certain value. 3) an arithmetic process for calculating the relational expression between the number of cycles in a nucleic acid sample of known concentration and the number of copies of the target nucleic acid at the start of the reaction; 4) the number of copies of the target nucleic acid in the unknown sample at the start of the PCR. Real-time quantitative PCR, comprising: Data analysis method for the law. 38. Perform PCR on the target nucleic acid using the nucleic acid probe according to any one of claims 2 to 11,
A method for analyzing a melting curve of a nucleic acid, comprising analyzing a melting curve of the nucleic acid with respect to the amplified nucleic acid to obtain a Tm value. 39. A nucleic acid characterized by performing PCR on a target nucleic acid using the nucleic acid probe according to claim 4 or 7 as a primer, and analyzing the melting curve of the amplified nucleic acid to determine a Tm value. Analysis method of melting curve. 40. The data analysis method for a real-time quantitative PCR method according to any one of claims 34 to 37, wherein the novel method for analyzing a melting curve of a nucleic acid according to claim 38 and / or 39 is used. Analyzing the melting curve of the nucleic acid, ie, gradually increasing the temperature of the nucleic acid amplified by the PCR method of the present invention from a low temperature until the nucleic acid is completely denatured. A process of measuring the intensity, a process of displaying the measurement results on a display every time (a process of displaying a melting curve of a nucleic acid), a process of first-order differentiation of the melting curve, and a value (−
dF / dT, F: fluorescence intensity, T: time) as a differential value on a display, and an inflection point from the differential value. Data analysis method. 41. A step of analyzing data by the data analysis method according to claim 34, a step of analyzing data by the data analysis method according to claim 35, and an analysis of data by the data analysis method according to claim 36. 41. A method for real-time quantitative PCR measurement and / or comprising: a step of analyzing data by the data analysis method according to claim 37; a step of analyzing data by the data analysis method according to claim 40. Or an analyzer. 42. A step of analyzing data by the data analysis method according to claim 34, a step of analyzing data by the data analysis method according to claim 35, and an analysis of data by the data analysis method according to claim 36. A computer-readable program storing a program for causing a computer to execute a step of performing data, a step of analyzing data by the data analysis method according to claim 37, and a step of analyzing data by the data analysis method according to claim 40. recoding media. 43. The method for quantifying a nucleic acid according to claim 34, wherein
37. Real-time quantitative P according to any one of 37 and 40
A method for quantifying a nucleic acid, comprising utilizing a data analysis method for a CR method. 44. A method for quantifying a nucleic acid, wherein the method according to claim 41 is used in a method for quantifying a nucleic acid. 45. A method for quantifying a nucleic acid, comprising using the computer-readable recording medium according to claim 42.