JP2002191372A - New nucleic acid probe, method for assaying nucleic acid and method for analyzing data obtained by the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new nucleic acid probe for simply and efficiently assaying a target nucleic acid or performing polymorphism analysis and real time quantitative PCR, or the like. SOLUTION: (1) The new probe has a hybridization reaction-based fluorescent intensity which reduces before and after the hybridization reaction when the nucleic acid probe is hybridized with a target nucleic acid. (2) The nucleic acid probe comprises an oligonucleotide in which, in order to increase the hybridization reaction-based fluorescent intensity before and after the hybridization reaction, a stem loop is not formed between a base chain at a site at which a fluorescent coloring matter and a quencher substance are labeled and the fluorescent coloring matter is labeled and at a site at which the quencher substance is labeled. The assay kit, the DNA chip, the nucleic acid assay method, the polymorphism analysis method and the real time quantitative PCR method use the above mechanism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fluorescent dye and / or a fluorescent dye.
And a novel nucleic acid probe labeled with a quencher substance. Specifically, when the nucleic acid probe is hybridized to the target nucleic acid, the fluorescence intensity of the hybridization reaction system increases or decreases,
A fluorescent dye and / or a quencher substance is labeled on a single-stranded oligonucleotide. Further, the present invention relates to a nucleic acid measurement method using the nucleic acid probe.
The present invention also relates to measurement kits and measurement devices used in the methods and various measurement devices related thereto. Also, the present invention relates to a method for analyzing species and amounts of various nucleic acids, a method for analyzing data obtained by those methods, and a computer-readable recording medium which records a procedure for causing a computer to execute a process of the analysis method as a program. .

[0002]

2. Description of the Related Art Conventionally, various methods for measuring a nucleic acid concentration 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 a nucleic acid probe labeled with a fluorescent dye are hybridized on a membrane, unreacted nucleic acid probes are washed away, and the nucleic acid probe hybridized with the target nucleic acid is labeled. This is for measuring the fluorescence intensity of only the fluorescent dye molecules thus obtained.

(2) Intercalator method (Glazer e
t 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, pp. 46-51, 1997)
Year, Yodosha), SYBR R Green (I)
^TM System; pamphlet issued by Roche Diagnostics, Inc. on April 5, 1999).

(3) FRET (fluorescence energy tr)
ansfer) (Mergney et al., Nucleic Acid
Res., 22: 920-928, 1994. This method consists in hybridizing two nucleic acid probes to a target nucleic acid. Each of the two nucleic acid probes is labeled with a different fluorescent dye. One fluorescent dye of the two probes can transmit energy to the fluorescent dye of the other probe to emit light through the FRET phenomenon. The two probes are designed so that the fluorescent dyes face each other and hybridize 1 to 9 bases apart. Therefore, when the two nucleic acid probes hybridize to the target nucleic acid, the latter fluorescent dye emits light, and the intensity of the light emission is proportional to the amount of replication of the target nucleic acid.

(4) Molecular beacon method (Tyagi et al.,
(Nature Biotech., 14: 303-308, 1996.) The nucleic acid probe used in this method has a nucleic acid probe labeled with a reporter dye at one end and a quencher dye at the other end. Since both ends are complementary to each other in the base sequence, the base sequence is designed so as to form a hairpin structure as a whole probe. When suspended in liquid due to its structure, the emission of the reporter dye is suppressed by the quencher dye due to Forster resonance energy.
However, when hybridized to the target nucleic acid, the hairpin structure is broken, and the distance between the reporter dye and the quencher dye is increased, so that transfer of Forster resonance energy does not occur. Therefore, the reporter dye emits light.

(5) Davis method (Davis et al., N
ucleic acids Res. 24: 702-706, 1996)
A probe was prepared in which a fluorescent dye was bound to the 3 ′ end of the oligonucleotide via a spacer having 18 carbon atoms. This was applied to flow cytometry. 3 '
It was reported that a 10-fold fluorescence intensity was obtained when hybridization was carried out as compared with the case where a fluorescent dye was directly bound to the end. These methods include various nucleic acid measurement methods, FISH method (fluorescent in situ hybridization assays), PC
It has been applied to R method, LCR method (ligase chain reaction), SD method (strand displacement assays), competitive hybridization method and the like, and has made remarkable development.

(6) PCR method (protein, nucleic acid, enzyme;
(Vol. 35, No. 17, 1990, Kyoritsu Publishing Co., Ltd.) (Experimental Medicine, Vol. 15, No. 7, 1997, Yodosha). In addition, the genes of various diseases, particularly, diseases relating to immunity have been elucidated, and some results have been obtained.

These methods are currently generally used. However, after performing a hybridization reaction between a nucleic acid probe labeled with a fluorescent dye and a target nucleic acid, it is necessary to wash the unhybridized nucleic acid probe from 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.

Further, in the conventional polymorphism analysis method, since the amplification of the gene is performed using PCR without quantification, the amount of the gene and the composition ratio of the polymorphism before the amplification of the target gene are increased. Could not be analyzed.

[0010]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a method for measuring the concentration of a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye in a shorter time.
An object of the present invention is to provide a method that can more simply and accurately measure the concentration of a target nucleic acid, nucleic acid probes used for the method, and various devices using the probes.

A second object of the present invention is to provide a novel polymorphism analysis method for simply and rapidly measuring the polymorphism composition ratio of a target gene, reagent kits used therefor, and quantitative polymorphism analysis. An object of the present invention is to provide a computer-readable recording medium storing a program of a procedure for causing a computer to execute a method of analyzing data obtained by the method, and an analyzer for quantitative polymorphism analysis.

[0012]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted various studies as described below.
The following findings were obtained. Various nucleic acid probes were examined in detail, and a large number of probes were produced by trial and error. As a result, even in the case of a nucleic acid probe consisting of an oligonucleotide that does not form a stem-loop structure between the base chain where the fluorescent dye is labeled and the base chain where the quencher substance is labeled, By labeling the substance, the quencher substance may act on the emission of the fluorescent dye, which may have a quenching effect on the emission.

[0013] A method for measuring the concentration of a nucleic acid using a nucleic acid probe was studied repeatedly. As a result, when the nucleic acid probe labeled with the fluorescent dye hybridizes to the target nucleic acid, there is a phenomenon (fluorescence quenching phenomenon) in which the emission of the fluorescent dye is reduced (fluorescence quenching phenomenon). In particular, the degree of the reduction depends on the type or base sequence of the base to which the fluorescent dye is bound.

After amplifying a target gene using a quantitative gene amplification method, polymorphism analysis is performed on the target gene, so that the amount of the target gene before amplification and the composition ratio of the polymorphism of the gene can be measured. It can be performed simply, quickly and with good quantitativeness. The present invention has been completed based on the above findings.

[0015]

That is, the present invention provides: 1) a nucleic acid probe comprising a single-stranded oligonucleotide capable of hybridizing to a target nucleic acid, and a fluorescent dye and a quencher substance being labeled; When the probe is hybridized to the target nucleic acid, a fluorescent dye and a quencher substance are labeled on the oligonucleotide so that the fluorescence intensity of the hybridization reaction system increases, and the oligonucleotide is labeled with a fluorescent dye. Novel nucleic acid probe for nucleic acid measurement, characterized in that there is no formation of a stem-loop structure between the base where the quencher substance is located and the quencher substance, or

2) The novel nucleic acid probe for measuring nucleic acid according to 1) above, wherein the fluorescent dye and the quencher substance are labeled at the same nucleotide position of the single-stranded oligonucleotide, or

3) The distance between the base where the fluorescent dye is labeled and the base where the quencher substance is labeled is 1 to 20 or ｛(any of 3 to 8) in terms of the number of bases. The novel nucleic acid probe for measuring a nucleic acid according to the above 1), wherein (integer) + 10n｝ (where n is an integer including 0),
Or 4) the novel nucleic acid probe for nucleic acid measurement according to any one of 1) to 3) above, wherein the single-stranded oligonucleotide has the same chain length as the target nucleic acid, or

5) A nucleic acid probe labeled with at least one fluorescent dye, wherein, when hybridized to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces its luminescence, and the probe is: 5 '
When the nucleic acid probe is labeled with the fluorescent dye at a portion other than the terminal phosphate group or the 3′-terminal OH group of ribose or deoxyribose at the 3 ′ end, and the nucleic acid probe hybridizes to the target nucleic acid, the modification is performed. The base sequence of the probe is designed so that one to three bases are separated from the base of the target portion (the base of the modified portion is counted as 1), and at least one base of G (guanine) is present in the base sequence of the target nucleic acid. When a nucleic acid probe labeled with a fluorescent dye hybridizes to a target nucleic acid, the fluorescent dye reduces its emission, and the nucleic acid labeled with a fluorescent dye for measuring the concentration of the target nucleic acid. Probes, also

6) A nucleic acid probe labeled with at least one fluorescent dye, wherein the fluorescent dye is a nucleic acid probe that reduces its luminescence when hybridized to a target nucleic acid, and the probe is: 5 '
A terminal phosphate group or is labeled with the fluorescent dye at a portion other than the 3′-position OH group of 3′-terminal ribose or deoxyribose, and when the nucleic acid probe is hybridized to the target nucleic acid, A plurality of base pairs of the probe-nucleic acid hybrid form at least one pair of G (guanine) and C (cytosine) at a distance of 1 to 3 bases from the base of the modified portion (the base of the modified portion is counted as 1). The base sequence of the probe is designed, and when the nucleic acid probe labeled with a fluorescent dye hybridizes to the target nucleic acid, the fluorescent dye reduces the emission of the target nucleic acid, A nucleic acid probe labeled with a fluorescent dye for concentration measurement,
Also,

7) When the 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,
In addition, the probe is labeled with the fluorescent dye at the phosphate group at the 5 ′ end and the OH group at the 3 ′ position of ribose or deoxyribose at the 3 ′ end, and the nucleic acid probe is attached to both ends. When the probe hybridizes to the target nucleic acid, the probe-nucleic acid hybrid has at least one base pair separated from the terminal base of the target nucleic acid hybridized to the probe by 1 to 3 bases (the terminal base is counted as 1). G
The base sequence of the probe is designed to form a pair of (guanine) and C (cytosine), and when the nucleic acid probe labeled with the fluorescent dye hybridizes to the target nucleic acid, the fluorescent dye A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid, characterized in that its emission is reduced,

8) The nucleic acid probe wherein the 3′-carbon hydroxyl group of 3′-terminal ribose or deoxyribose or the 3′- or 2′-carbon hydroxyl group of 3′-terminal ribose is phosphorylated. A) a nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to any one of the above, or 9) a phosphate group at the 5 ′ end and / or 3 ′ end of the nucleic acid probe labeled with a fluorescent dye The nucleic acid probe labeled with a fluorescent dye for measuring the concentration of the target nucleic acid according to any one of 1) to 7) above, or

10) The oligonucleotide of the nucleic acid probe for nucleic acid measurement, wherein the oligonucleotide is a chemically modified nucleic acid.
9) The nucleic acid probe labeled with the fluorescent dye for measuring the concentration of the target nucleic acid according to any one of the above 9), or 11) the chemically modified nucleic acid is a 2′- O -methyl oligonucleotide, 2′- O − Ethyl oligonucleotide,
The nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to the above 10), which is a 2′- O -butyl oligonucleotide, a 2′- O -ethylene oligonucleotide, or a 2′- O -benzyl oligonucleotide. Or

12) The oligonucleotide of the nucleic acid probe for nucleic acid measurement is a chimeric oligonucleotide containing ribonucleotides and deoxyribonucleotides.
any one of the above 1) to 11) which is an oligonucleotide)
Or a nucleic acid probe labeled with a fluorescent dye for measuring a concentration of a target nucleic acid according to 13), or 13) chimeric oligonucleotide is a 2′- O -methyl oligonucleotide, 2′- O -ethyl oligonucleotide, 2) '- O - butyl oligonucleotide or 2'O - benzyl fluorescent dye-labeled nucleic acid probe for density measurement of the target nucleic acid according to the 12) is an oligonucleotide intended to intervening or,

(14) A method for measuring the concentration of a target nucleic acid, comprising hybridizing the nucleic acid probe according to any one of the above (1) to (13) to a target nucleic acid and measuring the fluorescence intensity of a measuring system. 15) The target nucleic acid, wherein the nucleic acid probe for nucleic acid measurement according to any one of the above 1) to 13) is hybridized to a target nucleic acid, and the amount of change in emission of the fluorescent dye before and after hybridization is measured. Concentration measurement method, or

16) In the method of measuring the concentration of a target nucleic acid using the nucleic acid probe according to any one of the above 10) to 13), under conditions suitable for sufficiently destroying the higher-order structure of the target nucleic acid. A method for measuring the concentration of the target nucleic acid, which comprises subjecting the target nucleic acid to heat treatment and then hybridizing the nucleic acid probe and the target nucleic acid; or 17) a method for performing a hybridization reaction on a hybridization reaction system before the hybridization reaction. The method for measuring the concentration of a target nucleic acid according to 16), wherein a helper probe is added, or

18) In a method for analyzing or measuring polymorphism and / or mutation of a target nucleic acid, the nucleic acid probe according to any one of 1) to 13) above is hybridized to the target nucleic acid. A method for analyzing or measuring a polymorphism or / and mutation of a target nucleic acid, which comprises measuring a change in fluorescence intensity, or

19) Amplifying the target gene by a quantitative gene amplification method and analyzing the polymorphism of the target gene to measure the amount of the target gene, the composition ratio of the polymorphism of the gene or the amount of each component. Or 20) polymorphism analysis is performed by T-RFLP (terminal restriction).
fragment length polymorphism), RFLP (restric
Option fragment length polymorphism) method, SSCP
(Single strand conformation) method or CFLP
(Cleavage fragment length polymorphism) The quantitative polymorphism analysis method according to 19) above, or

21) When the quantitative gene amplification method is quantitative P
The quantitative polymorphism analysis method according to the above 19) or 20), which is a CR method, or 22) the quantitative PCR method according to any one of the above 1) to 13)
In the quantitative polymorphism analysis method according to the above item 21), wherein the nucleic acid probe according to any one of the above items 1) to 13) is used as a primer, The quantitative polymorphism analysis method according to 21), wherein the amount of change in the emission of the fluorescent dye is measured, or

24) The above-mentioned 21) to 23) wherein the quantitative PCR method is a real-time monitoring quantitative PCR method.
25) The method for quantitative polymorphism analysis according to any one of the above, or 25) a kit for measuring the concentration of a target nucleic acid, which contains or accompanies the nucleic acid probe according to any one of the above 1) to 13). A kit for measuring the concentration of the target nucleic acid, or

26) The kit for measuring the concentration of a target nucleic acid according to the above 25) containing or accompanying a helper probe, or 27) a measuring kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, A) a kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, which comprises the nucleic acid probe according to any one of) to 13); or

28) A measurement kit for analyzing or measuring the polymorphism and / or mutation of the target nucleic acid according to the above 27) containing or accompanying a helper probe, or 29) any of the above 19) to 24) A reagent kit for use in a quantitative PCR method in the quantitative polymorphism analysis method according to 1, wherein the reagent kit contains or is attached to the nucleic acid probe according to any one of the above 1) to 13). Characteristic quantitative PCR reagent kit, or

30) A plurality of the nucleic acid probes according to any one of the above 1) to 13) are bound to the surface of the solid support, and the target nucleic acid is hybridized to the corresponding nucleic acid probe. 31) a device for measuring the concentration of at least one target nucleic acid among a plurality of nucleic acids, wherein the concentration of the target nucleic acid can be measured by the measurement; or In the device for nucleic acid measurement according to the, nucleic acid probes are arranged in an array on the surface of the solid support,
A device for measuring the concentration of one or more nucleic acids bound, respectively, or

32) 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 so that the corresponding nucleic acid probe binding region is at the optimal temperature condition. A device for measuring the concentration of at least one target nucleic acid of the plurality of nucleic acids according to 30) or 31), which can be temperature-controlled, or

33) Measuring the concentration of at least one target nucleic acid among a plurality of nucleic acids, wherein the target nucleic acid is measured using the nucleic acid measuring device according to any one of 30) to 32). 34) A method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, characterized by using the nucleic acid measuring device according to any one of the above 30) to 32), or 35) 30. A quantitative polymorphism analysis method using the nucleic acid measurement device according to any one of 30) to 32), or

36) The target nucleic acid according to any one of the above 14) to 17), wherein the target nucleic acid is a nucleic acid contained in cells derived from microorganisms or animals obtained by pure isolation or contained in a homogenate of those cells. 37) the method for measuring the concentration of the target nucleic acid according to the above, or 37) the polymorphism of the target nucleic acid according to the above 18), wherein the target nucleic acid is a nucleic acid of an intracellular or homogenate of a cell of a complex microbial system or a symbiotic microbial system, or / And a method for analyzing or measuring the mutation, or

38) The method for quantitative polymorphism analysis according to any one of 19) to 24) above, wherein the target nucleic acid is a nucleic acid of intracellular or homogenate of a complex microorganism system or a symbiotic microorganism system. 39) In the PCR method, a reaction is carried out using the nucleic acid probe according to any one of 1) to 13) above, and amplification is performed based on a change rate of a fluorescence intensity value generated by hybridization between the probe and the amplified target nucleic acid. A method for measuring the concentration of a target nucleic acid used in a PCR method, which comprises measuring an initial concentration of a target nucleic acid, or

40) In the PCR method, 1) to 1
The reaction was performed using the nucleic acid probe according to any one of 3) as a primer, and amplification was performed based on a change rate of a fluorescence intensity value generated by hybridization of the primer or an amplified nucleic acid amplified from the primer with an amplified target nucleic acid. A method for measuring a target nucleic acid using a PCR method, which comprises measuring an initial concentration of the target nucleic acid, or

41) In the PCR method, 1) to 1
A reaction system in which a reaction is carried out using the nucleic acid probe according to any one of the above 3), and the probe is decomposed and removed by a polymerase during a nucleic acid extension reaction, or a reaction system during a nucleic acid denaturation reaction or when a nucleic acid denaturation reaction is completed Measuring the fluorescence intensity value and the fluorescence intensity value of the reaction system when the target nucleic acid or the amplified target nucleic acid is hybridized with the nucleic acid probe, and calculating the rate of change of the fluorescence intensity value from the former. A method for measuring an initial concentration of a target nucleic acid amplified by PCR, or

42) In the PCR method, 1) to 1
3) performing a reaction using the nucleic acid probe according to any one of the above as a primer, the fluorescence intensity value of the reaction system in which the probe and the target nucleic acid or the amplified target nucleic acid are not hybridized, and the nucleic acid probe being the target nucleic acid or the amplified target nucleic acid. A method for measuring the initial concentration of the target nucleic acid amplified by PCR, which comprises measuring the fluorescence intensity value of the reaction system when hybridizing, and calculating the reduction rate of the former fluorescence intensity value, or ,

43) The PCR method is real-time quantitative P
The method for measuring the initial concentration of a target nucleic acid amplified by PCR according to the above 41) or 42) which is a CR method, or 44) any one of the above 14) to 24), or the above 33)
-43) in the method for analyzing data obtained by the nucleic acid measurement method according to any one of (1) to (3), wherein the fluorescence intensity value of the reaction system after the target nucleic acid is hybridized with a nucleic acid probe labeled with a fluorescent dye, A data analysis method for a method for measuring the concentration of a target nucleic acid, which is corrected by the fluorescence intensity value of the reaction system obtained after the probe-nucleic acid hybrid complex formed as described above is dissociated, or

45) In the method for analyzing data obtained by the real-time quantitative PCR method described in 43) above, after the amplified nucleic acid in each cycle binds to the fluorescent dye, or the amplified nucleic acid is labeled with the fluorescent dye. The fluorescence intensity value of the reaction system after hybridization with the nucleic acid probe thus obtained is determined by the fluorescent dye-nucleic acid complex formed in each cycle or the probe thus formed.
Data analysis for a real-time quantitative PCR method, comprising an arithmetic processing step for correcting with a fluorescence intensity value of a reaction system obtained after dissociation of a nucleic acid hybrid complex (hereinafter referred to as a correction arithmetic processing step). Method, or

46) The data analysis method for the real-time quantitative PCR method according to 45), wherein the correction operation processing according to 45) is based on the following [Equation 1] or [Equation 2]. Or f _n = f _{hyb, n} / f _{den, n} [Formula 1] f _n = f _{den, n} / f _{hyb, n} [Formula 2] [where, f _n : [Formula 1] or [Formula 2] F _{hyb, n} : In the n-th cycle, after the amplified nucleic acid binds to the fluorescent dye or the amplified nucleic acid hybridizes with the nucleic acid probe labeled with the fluorescent dye, The fluorescence intensity value of the subsequent reaction system, f _{den, n} : the fluorescence intensity value of the reaction system after dissociation of the formed fluorescent dye-nucleic acid complex or the formed probe-nucleic acid hybrid complex in the nth cycle ]

47) Analyzing the data obtained by the real-time quantitative PCR method described in 43) above)
In each cycle,
Alternatively, the correction calculation processing value calculated by [Equation 2] is substituted into the following [Equation 3] or [Equation 4], and the fluorescence change ratio or the fluorescence change ratio between each sample in each cycle is calculated and compared. data analysis methods _{or, F n = f n / f} a [equation 3] F _n = f _a / f _n [equation 4] wherein, for real-time quantitative PCR method, which comprises, F _n : Fluorescence change rate or fluorescence change rate calculated by [Equation 3] or [Equation 4] in the nth cycle, f _n : Correction operation processing value by [Equation 1] or [Equation 2] f _a : [Equation 1] ] Or a correction calculation processing value according to [Equation 2] with an arbitrary number of cycles before a change in f _n is observed]

48) Analyzing the data obtained by the real-time quantitative PCR method described in 43) above 47)
In the method described in (1), using (5), (6) or (7), using the fluorescence change rate or the data of the fluorescence change rate calculated by (Formula 3) or (Formula 4), Calculation process, 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 and b are arbitrary numerical values, F _n : [Equation 3] or [Equation 4] ] (2) a calculation process for obtaining the number of cycles in which the calculation value of (1) has reached a fixed value, and (3) a nucleic acid of a known concentration. Calculation processing to calculate the relational expression between the number of cycles in the sample and the number of copies of the target nucleic acid at the start of the reaction , (4) data analysis method for real-time quantitative PCR method characterized by an arithmetic process, for obtaining the number of copies of the target nucleic acid at the PCR started in an unknown sample or,

49) Regarding the target nucleic acid 1) to 13)
PCR is performed using the nucleic acid probe according to any one of the above, and the melting curve of the target nucleic acid is analyzed to determine the T of each amplified nucleic acid.
a method for analyzing a melting curve of a target nucleic acid, characterized by obtaining an m value, or

50) The data analysis method for a real-time quantitative PCR method according to any one of the above 45) to 48), wherein the nucleic acid amplified by the PCR method is cooled from a low temperature until the nucleic acid is completely denatured. A process of gradually increasing the temperature, a process of measuring the fluorescence intensity at predetermined time intervals in this process, a process of displaying the measurement results on the display as a function of time and drawing a melting curve of the nucleic acid on the display, The differentiated value of the curve (-dF
/ DT, F: fluorescence intensity, T: time), displaying the differentiated value as a differential value on a display,
A data analysis method for a real-time quantitative PCR method, comprising a step of obtaining an inflection point from the differential value.

[0047]

Next, the present invention will be described in more detail with reference to preferred embodiments. The present invention consists of three inventions. A feature of the first invention is a nucleic acid probe obtained by labeling a single-stranded oligonucleotide that hybridizes to a target nucleic acid with a fluorescent dye and a quencher substance, wherein the nucleic acid probe is hybridized to the target nucleic acid. In some cases, a fluorescent dye and a quencher substance are labeled on the oligonucleotide, and a place where the fluorescent dye is labeled and a place where the quencher substance is labeled so that the fluorescence intensity of the hybridization reaction system is increased. A novel nucleic acid probe for nucleic acid measurement, characterized in that the oligonucleotide does not form a stem-loop structure between base chains. Therefore, hereinafter, for convenience, the nucleic acid probe of the present invention may be referred to as a fluorescent probe or the nucleic acid probe of the first invention.

Next, a second aspect of the present invention is a nucleic acid probe labeled with a fluorescent dye. When the nucleic acid probe hybridizes to a target nucleic acid, the emission of the fluorescent dye decreases before and after hybridization. Is to measure. Incidentally, the nucleic acid probe of the present invention may be referred to as a fluorescence quenching probe or the nucleic acid probe of the second invention for simplification. A feature of the third invention of the present invention relates to various uses of the above-mentioned fluorescent probe and fluorescent quenching probe.

The technical terms used in the present invention will be described below. In the present invention, a probe-nucleic acid hybrid complex refers to a complex (complex) in which a nucleic acid probe labeled with the fluorescent dye of the present invention is hybridized with a target nucleic acid. For the sake of simplicity, hereinafter, it is abbreviated as a nucleic acid hybrid complex. Further, the fluorescent dye-nucleic acid complex refers to a complex in which a fluorescent dye is bound to a target nucleic acid. For example, a double-stranded nucleic acid with an intercalator bound thereto can be mentioned.

Also, in the present invention, nucleic acid probe, hybridization, hybridization, stem-loop structure, quenching, quenching effect, DNA, RNA, cD
NA, mRNA, rRNA, XTPs, dXTPs, NTPs, dNTPs, nucleic acid probes, helper nucleic acid probes (or nucleic acid helper probes, or simply helper probes), intercalators, primers, annealing, extension reactions, heat denaturation reactions, nucleic acid melting curves, PCR, RT-PCR, RNA-primed PCR, St
retch PCR, reverse PCR, PCR using Alu sequence, multiplex PC
R, PCR using mixed primers, PCR using PNA,
Hybridization assay method (hybridization
assays), FISH (fluorescent in situ hybridizati
on assays), PCR (polymerase chain assay)
s), LCR method (ligase chain reaction), SD method (strand displacement assays), competitive hybridization method (competitive hybridization), D
NA chip, nucleic acid detection (gene detection) device, S
Terms such as NP (snip: single nucleotide substitution polymorphism) and complex microbial systems have the same meanings as those generally used at present in molecular biology, genetic engineering, microbial engineering and the like.

The “target gene” and “target nucleic acid” refer to a nucleic acid or gene to be measured or quantified or qualitatively detected for its abundance or concentration. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids may be mixed. For example, a complex microbial system (mixed system of RNA or gene DNA of multiple microorganisms) or a symbiotic microbial system (RNA of multiple animals and plants and / or multiple microorganisms)
Alternatively, the specific nucleic acid is used for measuring the concentration in a mixed system of gene DNA. If the target nucleic acid needs to be purified, it can be performed by a conventionally known method. For example,
It can be performed using a commercially available purification kit or the like. Specific examples of the above nucleic acids include DNA, RNA, PN
A, oligodeoxyribonucleotide (oligodeoxyribonucleotide)
nucleotides), oligoribonucleic acid (oligoribonu
cleotides), and chimera nucleic acids of the above-mentioned nucleic acids.

In the present invention, measuring the concentration of a target nucleic acid means that one or more nucleic acids in a measurement system are used.
This means quantifying the concentration, performing qualitative detection, simply detecting, or analyzing polymorphisms / mutations and the like. In the case of multiple types of nucleic acids, quantitative detection of multiple types of nucleic acids at the same time, simple detection of multiple types of nucleic acids at the same time, or simultaneous analysis of polymorphisms / mutations of multiple types of nucleic acids, etc. Naturally, it is within the technical scope of the present invention.

The target nucleic acid concentration measuring device includes various types of D
Refers to an NA chip. Specific examples thereof include various types of DNA chips. In the present invention, any type of DNA chip may be used as long as the nucleic acid probe of the present invention can be applied.

A method for measuring the concentration of a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye (hereinafter, simply referred to as the nucleic acid probe of the present invention or the probe of the present invention) (hereinafter, for simplicity, simply, The nucleic acid measurement method is referred to as a hybridization method (hybridization assa).
ys), FISH method (fluorescent in situ hybridizat
ion assays), PCR method (polymerase chain assay)
s), LCR method (ligase chain reaction), SD method (strand displacement assays), competitive hybridization method (competitive hybridization) and the like to measure the concentration of the target nucleic acid.

The description starts with the fluorescent probe. The characteristic of this probe is that when it does not hybridize to the target nucleic acid, the emission of the fluorescent dye is inhibited by the quencher substance, but when it hybridizes, the inhibition is released and the fluorescence intensity Is an increasing probe. In the present invention, the fluorescent dye is a kind of fluorescent dye generally used for measuring and detecting nucleic acids by labeling a nucleic acid probe. For example, fluorescein or derivatives thereof (eg, fluorescein isothiocyanate (FITC) or derivatives thereof), Alexa 488, Alexa 532, cy3, cy5, 6-
joe, EDANS, rhodamine 6G (R6G) or a derivative thereof (eg, tetramethylrhodamine (terameth
ylrhodamine (TMR), tetramethylrhodamine isothiocyanate
e) (TMRITC), x-rhodamine, x-rhodamine, Texas red, BODIPY FL (trade name; Molecular Probes, USA), BODIPY FL / C3 (trade name; Molecular Probes, USA), BODIPY FL / C6 (trade name; Molecular Probes, USA), BODIPY 5-FAM (BODIPY) Trade names: Molecular Probes, USA), BODI
PY) TMR (trade name; Molecular Probe)
Probes, USA) or a derivative thereof (eg, BODIPY TR (trade name); Molecular Probes, USA), BODIPY (BODIY)
PY) R6G (trade name; Molecular Probe)
Probes, Inc., USA), BODIPY 564 (trade name; Molecular Probes, USA), BODIPY 581 (trade name; Molecular Probes, USA) ) And the like. Among them, FITC, EDAN
S, Texas Red, 6-joe, TMR, Alexa 488, Alexa 53
2, BODIPY FL / C3, BODIPY FL / C
6 and the like are preferred, and EDANS, Texas Red, FITC, TMR, 6-joe, BODIPY FL / C3, and BODIPY FL / C6 are more preferred.

A quencher substance is a substance that acts on the fluorescent dye to suppress or quench its emission.
For example, Dabcyl, QSY7 (Molecular probe), QS
Y33 (Molecular probe), Ferrocene or its derivative, methyl viologen, N, N'-dimethyl-2,9-diazopy
renium and the like, preferably Dabcyl and the like. By labeling a fluorescent dye and a quencher substance at a specific position on the oligonucleotide as described above, the luminescence of the fluorescent dye is quenched by the quencher substance.

In the present invention, the nucleic acid probe of the present invention is formed so that a single nucleotide which does not form a stem-loop structure between the base chain where the fluorescent dye is labeled and the base chain where the quencher substance is labeled is formed. The oligonucleotide of the strand is defined as the oligonucleotide in the self-chain due to the complementarity of at least two or more base sequences between the place where the fluorescent dye is labeled and the place where the quencher substance is labeled. At 2
An oligonucleotide that forms a heavy chain and does not form a stem-loop structure.

When the nucleic acid probe of the present invention is hybridized to the target nucleic acid, a fluorescent dye and a quencher are labeled on the oligonucleotide of the present invention so that the fluorescence intensity of the hybridization reaction system increases. Can be performed as follows. The distance between the base where the fluorescent dye is labeled and the base where the quencher substance is labeled is zero in terms of the number of bases, i.e., the same nucleotide of the single-stranded oligonucleotide where the fluorescent dye and the quencher substance are single-stranded. Label at the site, or 1 to 20 bases, or ｛(any integer from 3 to 8) +1
0n｝ (where n is an integer including 0). Preferably, 10 is added to the position of the same nucleotide in the single-stranded oligonucleotide, or 4 to 8 or any number thereof. More preferably, it is at the same nucleotide position of a single-stranded oligonucleotide, or 4 to 8. Thus, it is preferable to label each substance on the oligonucleotide. However, the distance between bases strongly depends on the base sequence of the probe, the fluorescent dye and quencher used for modification, the length of the linker that links them to the oligonucleotide, and the like. Therefore, it is difficult to completely specify the base interval, and the above-mentioned base interval is only a general example, and there are many exceptions.

When labeling is performed at the same nucleotide position of a single-stranded oligonucleotide, one is labeled with a base and the other is labeled with a moiety other than a base, ie, a phosphate moiety, a ribose moiety or a deoxyribose moiety. It is preferred to do so. In this case, it is preferable to label the 3 'end or the 5' end.

Alternatively, the distance between the fluorescent dye and the base for labeling the quencher substance is 1 to 20,
Or {(any integer from 3 to 8) + 10n} (where n is an integer including 0), or preferably 4 to 8
Or any number in them plus 10;
More preferably, when it is 4 to 8, each substance may be labeled in the oligonucleotide chain, and one substance may be labeled at the 5 ′ end or 3 ′ end of the oligonucleotide, and the corresponding other substance may be labeled. The substance may be labeled in the chain. Preferably, a fluorescent dye or quencher substance is labeled at the 5 'end or 3' end of the oligonucleotide, and a corresponding quencher substance or fluorescent dye is labeled from the end to the base having the above-mentioned number of bases. In this case, when labeling at the 3 ′ end or 5 ′ end, a base, a phosphate, or a ribose or deoxyribose may be
It is preferable to label the phosphate moiety, ribose moiety or deoxyribose moiety, and more preferably to label the phosphate moiety. When labeling in a chain, it is preferable to label a base in the chain. In addition, when modifying to a base in each of the above cases, any modification can be made as long as it can be modified. And the pyrimidine base O
H group, amino group, methyl group, and N-position at the 2-position may be modified (ANALYTICAL BIOCHEMISTRY, Vol. 225, pp. 32-38, 1998).
Year).

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

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

The nucleic acid of the present invention in which a modified RNA such as 2-O-methyl oligoribonucleotide (hereinafter simply referred to as 2-O-Me-oligoribonucleotide) is interposed in an oligodeoxyribonucleotide. Preferred results are obtained when the probe is used mainly for measuring RNA, particularly rRNA. When measuring RNA using the nucleic acid probe of the present invention, before hybridizing with the probe, an RNA solution as a sample is subjected to 80 to 100 ° C, preferably 90 to 100 ° C, optimally 93 to 97 ° C. And 1-1
Heat treatment for 5 minutes, preferably 2 to 10 minutes, optimally 3 to 7 minutes to destroy the higher order structure of the RNA is suitable for improving the hybridization efficiency.

Further, in order to further increase the efficiency of hybridization of the nucleic acid probe of the present invention to a hybridization sequence region, a helper probe is used.
It is preferred to add e) to the hybridization reaction solution. In this case, the oligonucleotide of the helper probe may be an oligodeoxyribonucleotide or oligoribonucleotide, or may be an oligonucleotide subjected to the same chemical modification as described above. As an example of the above-mentioned oligonucleotide, (5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) as a forward type, and (5 ′) CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT (5 ′) as a backward type or a reverse ward type. 3 ')
Having a base sequence of Preferable examples of the chemically modified oligonucleotide include 2-O-alkyl oligoribonucleotides, particularly 2-O-Me oligoribonucleotides. When the nucleic acid probe of the present invention has a base chain of 35 bases or less, the use of a helper probe is particularly effective. When using the nucleic acid probe of the present invention having a length of more than 35 base chains, it may be sufficient to simply heat denature the target RNA. As described above, when the nucleic acid probe of the present invention is hybridized to RNA, the hybridization efficiency is increased.
The fluorescence intensity increases with the amount of
RNA can be measured up to 50 pM. Thus, the present invention is also a measurement kit for measuring the concentration of a target nucleic acid, which contains or accompanies the helper probe in a measurement kit for measuring the concentration of a target nucleic acid, which contains or accompanies the nucleic acid probe of the present invention.

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

The 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 FISH method, and the application range of the present invention will be narrowed. If it is less than 5, non-specific hybridization is likely to occur, resulting in a large measurement error.

The oligonucleotide of the nucleic acid probe of the present invention can be produced by a general method for producing an oligonucleotide. For example, chemical synthesis methods, plasmid vectors,
It can be produced by a microbial method using a phage vector or the like (Tetrahedron letters, Vol. 22, pp. 1859-1862, 1981).
Year; Nucleic acids Research, 14, 6227-6245, 19
1986). It is preferable to use a commercially available nucleic acid synthesizer (for example, ABI394 (manufactured by Perkin Elmer, USA)). In addition, since there are companies that undertake commercial commissioned synthesis, the easiest way is to design only the base sequence and request synthesis there. Examples of such a company include Takara (Japan), Espec Oligo, and the like.

For labeling the oligonucleotide with a fluorescent dye and a quencher substance, any of the conventionally known labeling methods can be used (Nature Biotech).
nology, 14, 303-308, 1996; Applied and Envi
ronmental Microbiology, 63, 1143-1147, 1997
Year; Nucleic acids Research, 24, 4532-4535, 19
96). For example, when a fluorescent dye or a quencher substance is bonded to the 5′-terminal, first, a linker or a spacer, for example, — (CH ₂ ) _n —SH or — (CH ₂₎ introducing the _n -NH _2. These transductants are commercially available and may be purchased commercially (Midland Certified Reagent Companion).
y)). In this case, n is 3 to 8, preferably 6, 7. This linker or spacer has a -SH group or-
The oligonucleotide can be labeled by reacting a fluorescent dye or quencher substance having reactivity with the NH ₂ group. The thus synthesized oligonucleotide labeled with a fluorescent dye can be purified by reverse phase chromatography or the like to obtain the nucleic acid probe of the present invention.

In order to attach a fluorescent dye or a quencher substance to the 3 ′ end of the oligonucleotide, a C OH group at the 2 ′ or 3 ′ position of ribose or a C OH group at the 3 ′ position of deoxyribose as a linker, for example, - (CH _{2) n} -NH ₂ or - (CH ₂₎ introducing _n -SH. Since these introduced products are also commercially available in the same manner as described above, commercially available products may be purchased (Midland Cervified Residential Company (Midland CE)
rtified Reagent Company)). Further, a phosphate group is introduced into the OH group, and as a linker at the OH group of the phosphate group, for example,-(CH ₂ ) _n -SH or-(CH ₂ ) _n -N
H ₂ is introduced. In these cases, n is 3-9, preferably 4-8. This linker can be labeled oligonucleotides by reacting a fluorescent dye or quencher substance reactive to the -NH ₂ group or -SH group.

When introducing this amino group, kit reagents (for example, Uni-link aminomodifier (manufactured by CLONTECH, USA), Fluo Reporter Kit F-
6082, F-6083, F-6084, F-10220 (all manufactured by Molecular Probes, USA)
It is convenient to use Then, a fluorescent dye molecule can be bound to the oligoribonucleotide according to a conventional method. It is also possible to introduce fluorescent dye molecules into the probe nucleic acid chain (ANALYTICAL BIOCHEMISTRY
225, pp. 32-38 (1998)).

The ribose, deoxyribose, phosphate, base, linker,
In the case where an amino group is introduced into a fluorescent dye or quencher substance to enhance their reactivity, kit reagents (for example, Uni-link aminomodifier (manufactured by CLONTECH, USA), Fluo Reporter Kit (FluoReporter Kit) ) F-
6082, F-6083, F-6084, F-10220 (all manufactured by Molecular Probes, USA))
It is convenient to use Then, a fluorescent dye and a quencher substance can be bound to the oligoribonucleotide according to a conventional method. In the above-mentioned synthesis, introduction of a protective group to each functional group and elimination of the protective group can be carried out by a commonly known method.

As described above, an oligonucleotide labeled with a fluorescent dye and a quencher substance can be synthesized, but the intermediate compound and the completed compound are purified by liquid chromatography such as gel filtration and reverse phase. Is good. Thus, the nucleic acid probe of the present invention is obtained.

As described above, the design of the nucleic acid probe of the present invention is simple because it is only necessary to label the oligonucleotide having the base sequence that hybridizes to the target nucleic acid with the fluorescent dye and the quencher. The nucleic acid probe of the present invention can be obtained by request synthesis in the same manner as oligonucleotide synthesis, as long as the probe can be designed.

Next, a fluorescence quenching probe according to the second invention of the present invention will be described. In the probe of the present invention, a specific fluorescent dye labeled on an oligonucleotide has G
This phenomenon has been achieved by the present inventors discovering for the first time in the world that the fluorescence intensity decreases (fluorescence quenches) when (guanine base) is present (Japanese Patent Application No. 2001-133529). The feature of the probe of the present invention is an oligonucleotide labeled with at least one or more fluorescent dyes and at least one or more sites, and when hybridized to a target nucleic acid, the fluorescence intensity is reduced, It has properties opposite to those of fluorescent probes. The fluorescent dye used for labeling the present probe may be the same as the fluorescent dye described above. When a plurality of fluorescent dyes are used for labeling, the fluorescent dyes may be the same or different. It is sufficient that the fluorescence intensity decreases when the probe of the present invention is hybridized to the target nucleic acid. The labeling site is a terminal portion, both terminal portions, and a site in a chain. In short, it is only necessary that the object of the present invention can be achieved. When a plurality of fluorescent dyes are used, the measurement sensitivity is remarkably increased as compared with the case where a single fluorescent dye is used. In addition, when the fluorescent dye is of a different type, since the measurement can be performed at different wavelengths, the reliability of various analyzes and data analysis is significantly improved. Oligonucleotide of the fluorescence quenching probe of the present invention to be hybridized to the target nucleic acid,
It is the same as the above-mentioned fluorescent probe. That is, it may be a chimeric oligonucleotide or a chemically modified one. They may be interposed in the chain.

Further, in order to further increase the efficiency of hybridization of the nucleic acid probe of the present invention to a hybridization sequence region, a helper probe is used.
The addition of e) to the hybridization reaction solution, the base sequence of the helper probe, the number of base chains, the use of chemically modified oligonucleotides, and the like are the same as those of the invention. Then, when hybridized to RNA, the hybridization efficiency is increased, so that the fluorescence intensity is reduced according to the amount of RNA in the reaction solution, and the RNA can be measured up to a final RNA concentration of about 150 pM.

RNA using the fluorescence quenching probe of the present invention
The heat denaturation treatment of the RNA and the addition treatment of the helper probe in the measurement method described above are the same as in the case of the above-described fluorescent probe. Thereby, also in the method of the present invention, the hybridization efficiency becomes substantially 100%, 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. The base sequence of the probe is not particularly limited as long as it specifically hybridizes to the target nucleic acid. Preferably, when a nucleic acid probe labeled with a fluorescent dye has hybridized to a target nucleic acid, (1) one to three bases apart from the fluorescent dye labeling part of the target nucleic acid hybridized to the probe (the labeling base is assumed to be 1) ), G (guanine) or C in the base sequence of the target nucleic acid.
So that (cytosine) exists at least one base or more,
A nucleotide sequence for which the nucleotide sequence of the probe is designed,
(2) One to three bases apart from the fluorescent dye labeling part of the target nucleic acid hybridized to the probe (the base of the labeling part is counted as 1), and G (guanine) is added to the base sequence of the target nucleic acid.
Or C (cytosine) has at least one base or more,
In addition, at least one base pair in the fluorescent dye-labeled portion of the probe-nucleic acid hybrid complex has G
A base whose nucleotide sequence of the probe is designed to form a pair of (guanine) and C (cytosine) 1 to 3 bases away from the fluorescent dye labeling part (the labeling part base is counted as 1). Sequence. (3)
In the fluorescent dye-labeled portion of the probe-nucleic acid hybrid complex, a pair of G (guanine) and C (cytosine) having at least one base pair is separated from the fluorescent dye-labeled portion by 1 to 3 bases (labeled base Is counted as 1.),
A base sequence for which the base sequence of the probe is designed to be formed, and the like can be mentioned.

More preferably, (4) the above (1),
In (2) and (3), there may be mentioned, for example, a base sequence in which the fluorescent dye-labeled portion of the probe-nucleic acid hybrid complex is C (cytosine) or G (guanine), and the base forms a GC pair.

From the base sequence of the target nucleic acid,
Alternatively, in the case of a probe whose 5 'end cannot be designed as G or C, 5'-guanylic acid or guanosine or 5'-cytidylic acid or cytidine is added to the 5' end of the oligonucleotide which is a primer designed from the base sequence of the target nucleic acid. , The object of the present invention can be suitably achieved. Even if 5'-guanylic acid or 5'-cytidylic acid is added to the 3 'end, the object of the present invention can be suitably achieved. Therefore, in the present invention, a nucleic acid probe designed such that the base at the 3 ′ or 5 ′ end is G or C is a probe designed from the base sequence of a target nucleic acid, and a 5 ′ end of the probe in question. A probe obtained by adding 5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine to a probe, and a probe obtained by adding 5′-guanylic acid or 5′-cytidylic acid to the 5 ′ end of the probe. Defined to include.

The nucleic acid probe of the present invention is as described above.
However, a more specific form is as follows. the above
(1) having a base sequence of (2), (3),
5 'end, 3' end, base in chain, phosphate bond
Part, ribose part or deoxyribose part,
At least one or more sites (single or multiple
Probe labeled with a fluorescent dye
Can be Note that the labeling site at the 5 ′ end is phosphorous.
Ribose or deoxy in the case of acid group or dephosphorization
OH group at 5'-position of siribose, 2 'of ribose or
An OH group at the 3′-position or H at the 2′-position of deoxyribose
Or an OH group at the 3′-position.
The labeling site at the 3 'end is 2' or 3 'of ribose.
OH group or H group at 2'-position of deoxyribose
Or an OH group at the 3′-position. the above
Among the probes of (a), (a)
5'-terminal phosphate group and / or 3'-terminal ribose
Or the above except for the 3′-position OH group of deoxyribose
Labeling at least one or more sites
Probe, (b) 5 ′ terminal phosphate group and 3 ′ terminal
OH at the 3'-position of terminal ribose or deoxyribose
A probe in which both groups are labeled, and the like.
You.

The method for producing the oligonucleotide of the nucleic acid probe of the present invention and the method for labeling the oligonucleotide with a fluorescent dye are the same as in the above-mentioned invention (ANALYTICAL B
IOCHEMISTRY 225, pp. 32-38 (1998)).

Although the nucleic acid probe of the present invention can be prepared as described above, the most preferable form of the probe is one in which the 3 ′ and / or 5 ′ end is labeled with a fluorescent dye, and The base is G or C. Further, at least one or more G or C in the chain is labeled with a fluorescent dye. Also, the former and the latter are combined. In these forms, the OH group at the 3′-carbon of the 3′-terminal ribose or deoxyribose is
Further, the OH group at the 2′-position carbon of the 3′-terminal ribose may be modified with a modifying group such as a phosphate group, without any limitation. In this case, in the quantitative PCR described below, it cannot be used as a primer for chain elongation, but can be used as a nucleic acid probe.

The third invention of the present invention is an invention utilizing the above-mentioned fluorescent probe and fluorescent quenching probe. 1) Measuring Kit and Measuring Devices The fluorescence emitting probe and the fluorescence quenching probe (both probes are referred to as nucleic acid probes of the present invention unless otherwise specified for simplicity) are hybridized to a target nucleic acid, The amount of change in fluorescence intensity before and after hybridization (in the case of a fluorescent probe, the amount of increase in fluorescence intensity, and in the case of a fluorescence quenching probe, the amount of decrease in fluorescence intensity) can be measured in a simple, accurate, and short time. Measurement. In addition, it becomes possible to measure RNA, which was conventionally difficult to measure.

Thus, the present invention is a measurement kit for measuring the concentration of a target nucleic acid, which contains or accompanies the nucleic acid probe of the present invention. Further, the present invention is also a measurement kit for measuring the concentration of a target nucleic acid, which is obtained by including and accompanying the above-mentioned helper probe of the present invention in the kit.

The nucleic acid probe of the present invention can be suitably used not only for nucleic acid measurement but also for a method for analyzing or measuring polymorphism and / or mutation of a target nucleic acid. In particular, device for measuring target nucleic acid concentration 標的 DNA
Chip (protein nucleic acid enzyme, 43 volumes, 2004-20
(Page 11, 1998)} to provide a more convenient device for measuring the concentration of a target nucleic acid. Further, a method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid using the device is a very convenient method. That is, particularly in the case of a fluorescence quenching probe, GC in the hybridization with the nucleic acid probe of the present invention.
The fluorescence intensity changes depending on whether or not a pair is formed. Thus, by hybridizing the nucleic acid probe of the present invention to the target nucleic acid and measuring the luminescence intensity, the polymorphism and / or mutation of the target nucleic acid can be analyzed or measured. The specific method is described in the following examples. In this case, the target nucleic acid may be an amplified product amplified by one of various nucleic acid amplification methods, or may be an extracted product. The target nucleic acid may be of any type. However, a guanine base or cytosine base may be present in the chain or at the terminal. If no guanine base or cytosine base is present in the chain or at the end, the fluorescence intensity does not decrease. Therefore, according to the method of the present invention, G → A, G ← A, C → T, C ← T, G
→ C, G ← C mutation or substitution, ie, single nucleotide polymorphism (si
polymorphisms such as nucleotide nucleotide polymorphism (SNP)) can be analyzed or measured. At present, polymorphism analysis is currently performed by determining the base sequence of a target nucleic acid using the Maxam-Gilbert method and the dideoxy method.

Then, by including the nucleic acid probe of the present invention in a measurement kit for analyzing or measuring the polymorphism and / or mutation of the target nucleic acid, it is suitable as a measurement kit for analyzing or measuring the polymorphism and / or mutation of the target nucleic acid. Can be used.

When analyzing data obtained by the method for analyzing or measuring the polymorphism and / or mutation of the target nucleic acid of the present invention, the fluorescence intensity of the reaction system when the target nucleic acid hybridizes with the nucleic acid probe of the present invention. Providing a process for correcting the value by 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 a polymorphism and / or mutation of a target nucleic acid.

A feature of the present invention is that, in a measuring device for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, the fluorescence intensity value of the reaction system when the target nucleic acid hybridizes with the nucleic acid probe of the present invention. And a means for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, comprising means for performing correction processing based on the fluorescence intensity value of the reaction system when the above-mentioned substances are not hybridized.

Further, a feature of the present invention is that when analyzing data obtained by a method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, the target nucleic acid when hybridized with the nucleic acid probe of the present invention is analyzed. A computer-readable recording medium on which a procedure for causing a computer to execute a process of correcting the fluorescence intensity value of the reaction system by the fluorescence intensity value of the reaction system when the above-described one is not hybridized is recorded as a program. is there.

The nucleic acid probe of the present invention may be immobilized on a solid (support layer) surface, for example, a slide glass surface. In this case, it is preferable that the end not labeled with the fluorescent dye is fixed. This format is currently called a DNA chip. Monitoring of gene expression, determination of base sequence, mutation analysis (muta)
Option analysis) single nucleotide polymorphism
morphism (SNP)) and other polymorphism analyses (polymorphism analys
is). Of course, it can also be used as a nucleic acid measurement device (chip).

For binding the nucleic acid probe of the present invention to, for example, the surface of a slide glass, a slide glass coated with a polycation such as polylysine, polyethyleneimine or polyalkylamine, a slide glass into which an aldehyde group has been introduced, or an amino group can be used. First, the introduced slide glass is prepared. And, i) react the phosphate group of the probe with the slide glass coated with the polycation, ii)
An aldehyde group-introduced glass slide is reacted with an amino group-introduced probe. Iii) An amino group-introduced glass slide is subjected to PDC (pyridinium dichroma).
ate) introduction, and reaction with an amino or aldehyde group-introduced probe, 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 the nucleic acid probes of the present invention are arrayed and bound on a solid surface in an array form makes nucleic acid measurement more convenient. In this case, many types of target nucleic acids can be measured at the same time by creating a device in which many nucleic acid probes having different base sequences are individually bonded 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 surface to which the solid nucleic acid probe of the present invention is bonded, and the region of the solid to which the probe is bonded is set to an optimum temperature condition. Preferably, it is designed so that the temperature can be adjusted so that

In this device, a probe other than the nucleic acid 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 that is quenched or emits light due to the interaction, but has a structure designed to emit or quench light when hybridized to a target nucleic acid, that is, the aforementioned molecular beacon (Tyagi et al.)
al., Nature Biotech., 14: 303-308, 1996). 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, or rRNA is simply placed and hybridized. As a result, the amount of fluorescence changes in accordance with the amount of the target nucleic acid, and the target nucleic acid can be measured from the amount of change in the fluorescence. Further, by binding a large number of nucleic acid probes of the present invention having different base sequences on one solid surface, the concentration of many target nucleic acids can be measured at once. Therefore, it is a novel DNA chip because it can be used for measuring the concentration of a target nucleic acid in exactly the same application as the DNA chip. Under optimal reaction conditions, nucleic acids other than the target nucleic acid do not change the amount of fluorescence emitted, so that there is no need to perform an operation to wash unreacted nucleic acids. Further, by controlling the temperature of each nucleic acid probe of the present invention with a micro heater, it is possible to control the reaction conditions to the optimum for each probe, so that accurate measurement of the concentration becomes possible. Also,
The dissociation curve between the nucleic acid probe of the present invention and the target nucleic acid can be analyzed by continuously changing the temperature with a micro heater and measuring the amount of fluorescence during that time. From the difference in the dissociation curves, the properties of the hybridized nucleic acid can be determined, and the SNP can be detected. In addition, the present device also enables simultaneous gene amplification and gene detection by PCR or the like.

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

[0096] Although a large number of various probes are spotted on the device, the optimal hybridization conditions for nucleic acids hybridizing to each probe, such as the temperature, are different. Therefore, the hybridization reaction, the probe,
It is necessary to perform the washing operation under optimal conditions. However, since it is physically impossible, hybridization is performed at the same temperature for all probes, and washing is also performed at the same temperature with the same washing liquid. Therefore, the nucleic acid expected to be hybridized has a drawback that it does not hybridize, and even if it hybridizes, it is easily washed away because the hybridization is not strong. Due to such reasons, the quantification of nucleic acids was low. The present invention does not have such a disadvantage because this washing operation is not required. Further, by providing a micro heater at the bottom of the spot and controlling the hybridization temperature, a hybridization reaction can be performed at an optimum temperature for each probe of the present invention. Therefore, in the present invention, the quantitativeness is significantly improved.

2) Method for Measuring Target Nucleic Acid In the present invention, the concentration of the target nucleic acid can be measured in a short time, simply and specifically by using the above-described nucleic acid probe, measuring kit or device of the present invention. Can be. The measurement method is described below. In the measurement method of the present invention, first, the nucleic acid probe of the present invention is added to a measurement system and hybridized to a target nucleic acid. The method can be performed by a commonly known method (Analytical Biochemistr
y, 183, 231-244, 1989; Nature Biotechnolog
y, 14, 303-308, 1996; Applied and Environme
ntal Microbiology, 63, 1143-1147, 1997). For example, the hybridization conditions are as follows:
2 molar, preferably 0.1-1.0 molar, pH
Is 6 to 8, preferably 6.5 to 7.5.

[0098] The reaction temperature is preferably within the range of the Tm value of the nucleic acid hybrid complex obtained by hybridizing the nucleic acid probe of the present invention to a specific site of the target nucleic acid ± 10 ° C. By doing so, non-specific hybridization can be prevented. Tm-10
When the temperature is lower than 0 ° C, nonspecific hybridization occurs. When the temperature exceeds Tm + 10 ° C, no hybridization occurs. The Tm value can be determined in the same manner as in the experiment necessary for designing the nucleic acid probe of the present invention. That is, it hybridizes with the nucleic acid probe of the present invention (of a base sequence complementary to the nucleic acid probe).
Oligonucleotides are chemically synthesized using the above-described nucleic acid synthesizer or the like, and the T
The m value is measured in the usual way.

The reaction time is 1 second to 180 minutes, preferably 5 seconds to 90 minutes. When the time is shorter than 1 second, the number of unreacted nucleic acid probes of the present invention in hybridization increases. Further, it is meaningless if the reaction time is too long. The reaction time is greatly affected by the nucleic acid species, that is, the length of the nucleic acid or the base sequence. As described above, the nucleic acid probe of the present invention is hybridized to the target nucleic acid. Then, before and after the hybridization, the amount of luminescence of the fluorescent dye is measured with a fluorometer, and the amount of change in luminescence is calculated. Since the magnitude of the decrease is proportional to the concentration of the target nucleic acid, the concentration of the target nucleic acid can be determined.

The concentration of the target nucleic acid in the reaction solution: 0.1 to 10.0 nM
It is preferred that The concentration of the nucleic acid probe of the present invention in the reaction solution is preferably 1.0 to 25.0 nM. When preparing a calibration curve, it is desirable to use the nucleic acid probe of the present invention at a ratio of 1.0 to 2.5 with respect to the target nucleic acid.

Actually, 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 corresponding nucleic acid probes of the present invention are added to the sample, and the amount of change in the fluorescence intensity value is measured.
Then, the probe concentration corresponding to the largest one of the measured change values of the fluorescence intensity is set as a preferable probe concentration. The quantitative value of the target nucleic acid is determined from the calibration curve based on the amount of change in the fluorescence intensity measured with the probe having a preferable concentration.

Although the principle of the nucleic acid measurement method of the present invention is as described above, the present invention relates to various nucleic acid measurement methods such as FI
The method can be applied to the SH method, PCR method, LCR method, SD method, competitive hybridization method, TAS method, and the like.

For example, it is as follows. Application to FISH Method The method of the present invention can be applied to nucleic acids in microorganisms, plants and animals, and in cell homogenates. In addition, various types of microorganisms are mixed, or one or more types of microorganisms are mixed with cells derived from animals or plants, and cannot be isolated from each other in cells of a microbial system (complex microbial system, symbiotic microbial system). Alternatively, the method 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, and others, mycoplasmas, viruses, rickets, and the like can be mentioned. And the target nucleic acid in this system is, in these microbial systems,
For example, it refers to a nucleic acid having a nucleotide sequence having specificity for cells of a strain for which it is desired to examine how it is active. For example, 5S rRNA, 16S rRNA of a specific strain
Or a specific sequence of 23S rRNA or their gene DNA.

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

The above measuring method is, for example, as follows.
is there. That is, before adding the nucleic acid probe of the present invention,
Pre-set the temperature, salt concentration and pH of the combined or symbiotic microbial system
Adjust to the conditions described above. Complex or symbiotic microbial systems
The specific strain in⁷~Ten¹²Pcs / mL, good
Preferably 10⁹~Ten^TenIt is preferable to adjust to
You. It may be performed by dilution or concentration by centrifugation, etc.
Can be. 10 cells ⁷If the number is less than
The degree is weak and the measurement error increases. Ten¹²More than individual / mL
When the fluorescence intensity of a complex or symbiotic microorganism system is high,
Measurement of the quantity of specific microorganisms
And cannot do it. However, this range is the fluorescent light used
Depends on the performance of the meter.

The concentration of the nucleic acid probe of the present invention to be added is as follows:
It depends on the cell number of a particular strain in a complex or commensal microbial system. The concentration is 0.1 to 10.0 nM, preferably 0.5 to 5 nM, more preferably 1.0 nM per 1 × 10 ⁸ cells / mL. If it 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 of the present invention cannot be determined unconditionally because it depends on the amount of the target nucleic acid in the cell.

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

In the present invention, the components other than the microorganism in the complex microbial system or the symbiotic microbial system are the nucleic acid probe of the present invention and 5S rRNA or 16S rRNA of the specific strain.
Alternatively, it is not particularly limited as long as it does not inhibit hybridization with 23S rRNA or their gene DNA, and does not inhibit the emission of a fluorescent dye labeled on the oligonucleotide or the action of a quencher substance. For _{example, KH 2 PO 4, K 2} HPO 4, NaH 2 PO 4, phosphate salts such as Na ₂ HPO _4, ammonium sulfate, ammonium nitrate, inorganic nitrogen such as urea, magnesium, sodium, potassium, metal ions such as calcium ions , Various salts such as sulfates, hydrochloric acid, and carbonates of trace metal ions such as manganese, zinc, iron, and cobalt ions, and vitamins. If the above-mentioned inhibition is observed, the cells may be separated from a 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.

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 be used as the above buffer. . The concentration of the buffer is a concentration that does not inhibit hybridization and emission of the fluorescent dye.
The concentration depends on the type of buffer. Buffer pH is 4
-12, preferably 5-9.

Application to PCR Method Although any method can be applied as long as it is a PCR method, the case where it is applied to a real-time quantitative PCR method is described below. That is, in the real-time quantitative PCR method, PCR is performed using the specific nucleic acid probe of the present invention, and the amount of change in luminescence 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
It also includes PCR, inverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, PCR using PNA, and the like. Further, the term “quantitative” means quantitative measurement of the degree of detection in addition to the original quantitative measurement, as described above.

As described above, a target nucleic acid refers to a nucleic acid whose abundance is to be measured. With or without purification. Also, the magnitude of the concentration does not matter. Various nucleic acids may be mixed. For example, a specific nucleic acid to be amplified in a complex microbial system (mixed system of RNA or gene DNA of multiple microorganisms) or a symbiotic microbial system (mixed system of RNA or gene DNA of multiple animals and plants and / or multiple microorganisms).
If the target nucleic acid needs to be purified, it can be performed by a conventionally known method. For example, the purification 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 lowered and increased, and the increase in the emission of the fluorescent dye during the amplification process is monitored in real time (Experimental Medicine, Vol. 15, No. 7, No. 4,
6-51, 1997, Youtosha).

In the quantitative PCR method of the present invention, the target nucleic acid is amplified using the nucleic acid probe of the present invention, and the amount of change in the emission of the fluorescent dye during the amplification process, particularly, the increase in the fluorescence intensity in the case of the fluorescent probe. In the case of a fluorescence quenching probe, the amount of decrease in fluorescence intensity is measured. In the quantitative PCR of the present invention, the preferable nucleic acid probe of the present invention has 5 to 50 bases, preferably 10 to 25, and particularly preferably 15 to 20 bases.
In this regard, any compound may be used as long as it hybridizes 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, in the case of a fluorescent probe, the following can be mentioned. Any of the above-described fluorescent emission probes can be used,
Most preferred are those that are unlabeled at the 3 'end. That is, since the probe is used as a primer, the amount of a fluorescent dye, i.e., a target nucleic acid labeled with a quencher substance and a fluorescent dye, increases with the number of reactions. This is because the fluorescence intensity increases. Of course, those labeled at the 3 'end can be used sufficiently. In this case, it can be used as a simple nucleic acid probe.

In the case of a fluorescence quenching probe, all of the above-described fluorescence quenching probes of the present invention can be suitably used.

In particular, among the fluorescence quenching probes described above,
The nucleic acid probe of the present invention in which the 3 ′ terminal ribose or deoxyribose is labeled with a phosphate group or a fluorescent dye is designed so as not to be used as a primer. Real-time quantitative PCR using FRET phenomenon
PCR is performed using one nucleic acid probe of the present invention instead of two probes (labeled with a fluorescent dye) used in the method. The probe is added to the PCR reaction system, and PCR is performed. During the nucleic acid extension reaction, the probe hybridized to the target nucleic acid or the amplified target nucleic acid is decomposed by the polymerase and decomposed and removed from the nucleic acid hybrid complex. At this time, the fluorescence intensity value of the reaction system or the reaction system in which the nucleic acid denaturation reaction is completed is measured. Also, the fluorescence of a reaction system in which the target nucleic acid or the amplified target nucleic acid is hybridized with the probe (a reaction system during an annealing reaction or a nucleic acid extension reaction until the probe is removed from the nucleic acid hybrid complex by a polymerase). Measure the intensity value. Then, the amplified nucleic acid is measured by calculating the decrease rate of the fluorescence intensity value from the former. Fluorescence when the probe is completely dissociated from the target nucleic acid or amplified target nucleic acid by a nucleic acid denaturation reaction or when degraded and removed from the nucleic acid hybrid complex of the probe and the target nucleic acid or amplified target nucleic acid by polymerase during nucleic acid extension The intensity values are large.
However, a nucleic acid hybrid complex of the probe and the target nucleic acid or the amplified target nucleic acid by a polymerase during an annealing reaction or a nucleic acid extension reaction in which the annealing reaction has been completed or the probe is sufficiently hybridized to the target nucleic acid or the amplified target nucleic acid The fluorescence intensity value of the reaction system until it is decomposed and removed from is lower than the former. The decrease in the fluorescence intensity value is proportional to the amount of the amplified nucleic acid.

In this case, the Tm of the nucleic acid hybrid complex when the probe hybridizes with the target nucleic acid
Is the Tm value of the nucleic acid hybrid complex of the primer ±
It is desirable that the nucleotide sequence of the probe is designed to be in the range of 15 ° C., preferably ± 5 ° C.

Among the above-mentioned fluorescent quenching probes, those other than the probe in which ribose or deoxyribose at the 3 ′ end is labeled with a phosphate group or a fluorescent dye are added to the PCR reaction system as primers.
A PCR method using a primer labeled with a fluorescent dye has not yet been known except for the present invention. As the PCR reaction proceeds, the amplified nucleic acid is secondarily labeled with a fluorescent dye useful for practicing the present invention. Thus, the fluorescence intensity value of the reaction system in which the nucleic acid denaturation reaction is completed is large, but the fluorescence intensity of the reaction system is lower than that of the former in the case of annealing reaction completion or in the reaction system at the time of nucleic acid extension reaction. I do.

[0119] The PCR reaction can be carried out under the same reaction conditions as in the ordinary PCR method. Thus, the target nucleic acid can be amplified in a reaction system in which the Mg ion concentration is low (1-2 mM). Of course, the present invention can also be carried out in a reaction system in the presence of a high concentration (2 to 4 mM) of Mg ions used in conventionally known quantitative PCR.

In the PCR method of the present invention, the Tm value can be determined by performing the PCR of the present invention and analyzing the melting curve of the nucleic acid 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 of the present invention used as a primer can be suitably used. In this case, the nucleotide sequence of the nucleic acid probe of the present invention is represented by S
By making the sequence complementary to the region containing NP (snip; single nucleotide substitution polymorphism), after the PCR is completed, the dissociation curve of the nucleic acid is analyzed from the nucleic acid probe of the present invention. SNP can be detected.

3) Data Analysis Method One of the third inventions is an 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 record in which each procedure of the data analysis method is programmed and recorded. Medium (Also known as Sequence Detection Software S
ystem) and 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 Sequence Detection System (Sequence Detection Sy
stem SDS 7700) (Perkin Elmer Applied)
Biosystems (Perkin Elmer Applied Biosytems)
Company, USA)), the LightCycler ^TM system (Roche
Diagnostics Co., Germany) and the like are particularly preferable.

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

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

The PCR-related invention such as the data analysis method described above is an invention for analyzing data obtained by the above-described real-time quantitative PCR method. Each feature is described below. The first feature is that, in a method for analyzing data obtained by a real-time quantitative PCR method, when the amplified nucleic acid in each cycle binds to a fluorescent dye, or the amplified nucleic acid hybridizes with the nucleic acid probe of the present invention. The fluorescence intensity value of the reaction system at the time, the fluorescent dye and nucleic acid bound in each cycle, that is, the fluorescent dye-nucleic acid complex, or the nucleic acid probe of the present invention and the target nucleic acid hybridized, that is, nucleic acid This is an arithmetic processing step of correcting with the fluorescence intensity value of the reaction system when the hybrid complex is dissociated, that is, a correction arithmetic processing step.

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

As the correction calculation process in the correction calculation process, any process may be used 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 system when the amplified nucleic acid binds to the fluorescent dye or when the amplified nucleic acid hybridizes with the nucleic acid probe of the present invention in the nth cycle _{Fden, n} : the fluorescence intensity value of the reaction system when the above-described fluorescent dye-nucleic acid complex or nucleic acid hybrid complex is dissociated in the nth cycle] It includes a sub-step of displaying the corrected arithmetic processing value obtained in the processing on a computer display and / or similarly displaying and / or printing the value in the form of a graph as a function of the number of cycles.

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

F _n = f _n / f _a [Equation 3] F _n = f _a / f _n [Equation 4] [where, F _n : calculated by [Equation 3] or [Equation 4] in the nth cycle. Calculated fluorescence change rate or fluorescence change rate, f _n : correction calculation processing value by [Equation 1] or [Equation 2] in the nth cycle f _a : correction calculation processing value by [Equation 1] or [Equation 2] f _n is any number of cycles before the change is observed, but is usually, for example, 10 to 40 cycles, preferably 15 to 30 cycles, more preferably 20 to 30 cycles. Is adopted. In this process, the calculated value obtained in the above process is displayed and / or printed on a computer display.
Or a sub-step of similarly displaying and / or printing the comparison value or the value in the form of a graph as a function of the number of cycles, but with [Equation 1] or [Equation 2]
The above sub-steps may or may not be applied to the correction calculation processing value by.

The third feature is that (1) using the fluorescence change ratio or the data of the fluorescence change ratio calculated by [Equation 3] or [Equation 4], [Equation 5], [Equation 6] or [Equation 6] 7] log _b (F _n ), ln (F _n ) [Equation 5] log _b {(1-F _n ) × A}, ln {(1-F _n ) × A} [Equation 5] 6] log _b {(F _n -1) × A}, ln {(F _n -1) × A} [Equation 7]

[Wherein A and b: arbitrary numerical values, preferably integer values, more preferably natural numbers. When A = 100 and b = 10, {(F _n −1) × A} is expressed as 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]; (3) calculating the relational expression between the cycle number of the nucleic acid sample of known concentration and the copy number of the target nucleic acid at the start of the reaction, and (4) calculating the copy number of the target nucleic acid at the start of the PCR in the unknown sample. This is a data analysis method having an arithmetic processing step to be sought. Then, a process consisting of (1) → (2) → (3) → (4) is preferred.

In each of the above steps (1) to (3), the operation value obtained in each process is displayed on a computer display and / or the value is displayed in the form of a graph as a function of the number of cycles. Similarly, it may include a sub-step of displaying and / or printing. Since the operation processing value obtained in the process (4) needs to be printed at least, the process includes a sub-step of printing. The operation processing value obtained in the above (4) may be further displayed on a display of a computer. In addition, 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 displayed on a computer display in the form of a graph as a function of the number of cycles. Since they may or may not be printed, their display and / or printing sub-steps may be added as needed.

The fourth feature is that 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 steps of the data analysis method for analyzing the PCR using the analyzer of the present invention are programmed, and the computer executes the respective steps of the data analysis method of the present invention on a computer-readable recording medium on which the program is recorded. Is a computer-readable recording medium that records a program that can be executed.

A sixth feature is that, in the method for measuring nucleic acids, 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. Further, a seventh feature is that the method for analyzing the melting curve of the nucleic acid of the present invention, that is, the Tm of the nucleic acid by performing the PCR method of the present invention.
This is a method of analyzing data obtained by a method of obtaining a value. That is, for a 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, from 50 ° C to 95 ° C),
In this process, a short time interval (for example, 0.2 ° C. to 0.5
The process of measuring the fluorescence intensity at intervals corresponding to the temperature rise of ℃, the process of displaying the measurement results on the display as a function of time, ie, the process of displaying the melting curve of nucleic acids, and the differentiation of this melting curve by differentiation. This analysis method consists of obtaining the value (-dF / dT, F: fluorescence intensity, T: time), displaying the value as a derivative on the display, and finding the inflection point from the derivative. . In the present invention, at the time of nucleic acid extension reaction in each cycle, preferably, by adding to the above-described process, a process of performing an arithmetic 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.

An apparatus for measuring and / or 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 data analysis method of the novel PCR method of the present invention. It is within the technical scope of the present invention.

Furthermore, 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 procedure 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 records a program that enables a computer to execute the respective steps 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 procedure 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 Also referred to as a complex microbial system or a symbiotic microbial system, a microbial system in which various types of microorganisms are mixed or at least one type of microorganism is mixed with cells derived from other animals or plants and cannot be mutually isolated. It can be suitably used. The microorganism referred to here is a microorganism generally referred to, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms, other mycoplasmas,
Viruses, rickets and the like can be mentioned.

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

4) Polymorphism Analysis Method A feature of the method is that a nucleic acid is measured using the nucleic acid probe of the present invention in the polymorphism analysis method. In the present invention, “polymorphism” refers to biological polymorphisms. In the present invention, in particular, polymorphism of a gene (RNA, DNA, gene) that causes the polymorphism is considered. It is. It has the same meaning as currently used in molecular biology. “Polymorphism analysis” refers to analyzing and analyzing what polymorphisms are present in a gene.

Currently, the above polymorphism analysis methods include SSOP (seq
uence specific oligonucleotide probe) method, RFLP (re
striction fragment length polymorphism) method, T-RFL
P (terminal restriction fragment length polymorphis
m) method, SSCP (single strand conformation) method, MP
H method, CFLP (cleavase fragment length polymorphis
m) method, SSP (ssequences specific primer) method, PHFA
(preferential homoduplex formation formation assa
y) method, SBT (sequence based typing) method (PCR method, User's Guide, Chugai Medical Co., 1998;
Nucleic acid / enzyme: 35, 17, 1990, Kyoritsu Shuppan Co., Ltd .; Experimental Medicine, 15, 7 (extra number), 1997
In the present invention, all methods currently used for polymorphism analysis can be used, but in particular, T-RFLP
The method or the CFLP method can be suitably used.

Hereinafter, a specific description will be given step by step. The first feature resides in a method for quantitatively amplifying a gene using the nucleic acid probe of the present invention. As the method of quantitative gene amplification, any method having a quantitative property can be adopted. For example, PC
The R method can be suitably adopted. Among them, the quantitative PCR method or the real-time monitoring quantitative PCR method is a more preferable method. Conventionally known quantitative PCR methods include, for example, RT-PCR, RNA-primed PCR, Stretch PC
Examples of such methods include R, reverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, and PCR using PNA.

[0143] These conventionally known quantitative PCR methods include d
ATP, dGTP, dCTP and dTTP or d
Using UTP, a target gene (DNA or RNA), Taq polymerase, primers, and a nucleic acid probe or intercalator labeled with a fluorescent dye, Mg
In the presence of ions, the target gene is amplified while repeating low and high temperatures, and the increase in the emission of the fluorescent dye during the amplification process is monitored in real time (Experimental Medicine, Vol. 15, No. 7, No. 46- 51, 1997, Yodosha).

The quantitative PCR method using the nucleic acid probe of the present invention is a method using a probe labeled with a fluorescent dye. When the probe hybridizes with a target gene, the amount of emission of the fluorescent dye changes. (In particular, it increases in the case of a fluorescent probe, but decreases in the case of a fluorescent quenching probe.) This is a quantitative PCR method using a probe designed to be used. Specifically, using the nucleic acid probe of the present invention described in detail in the first invention and the second invention, the quantitative PCR described in detail in the third invention is used.
The gene amplification may be performed by the method of performing the method.

The gene amplification by the quantitative PCR method of the present invention can be achieved under conventionally known amplification reaction conditions. Amplification is preferably performed up to a commonly used amplification degree. In the process of amplifying the target gene, the fluorescence intensity value is measured with a fluorometer. The amount of change is proportional to the amount of the amplified gene. When plotting the change in the fluorescence intensity value as a function of time (cycle number in the case of the PCR method) on a normal graph, an S-shaped (sigmoid) curve is obtained. To a curve that increases linearly to a gentle plateau.

The degree of amplification of the target gene for improving the quantification of the initial gene amount before the start of PCR, that is, the time at which the gene amplification reaction is stopped depends on the purpose of polymorphism analysis, and is therefore particularly limited. Not something. That is, when only the preferential polymorphism of the polymorphism system is analyzed, it is preferable to amplify the target gene for an arbitrary time from when the change in fluorescence starts to be observed until the plateau is reached. Most preferably, the reaction is stopped at an exponential amplification phase (before reaching the midpoint of the sigmoid curve (point before the derivative of the curve becomes zero)). However, when analyzing all the polymorphisms included in the polymorphism system, several experiments are performed by trial and error to determine the amplification degree considered to be the best, and all the genes showing the polymorphism in the reaction system can be observed. It is good to amplify to the extent. Also, the amplification was divided into multiple times, that is, experiments were performed with multiple amplification degrees,
A method of comprehensively analyzing the results is also suitably adopted. This is because minor polymorphisms begin to draw sigmoid curves with large lags (delays) in time.

[0147] When a quantitative PCR method, particularly a real-time monitoring quantitative PCR method, is performed using the fluorescent quenching probe of the present invention as a primer, the fluorescent quenching probe as a primer is used one after another for amplification of the target gene. It is used to amplify a target gene labeled with a fluorescent dye. Then, the amplified target genes hybridize with the corresponding target genes. Upon hybridization, the fluorescence intensity value decreases. Then, the amplification reaction may be performed up to the optimum amplification degree in the same manner as described above by tracing the amount of decrease in the fluorescence intensity. The quantitative PCR
The reaction can be carried out under the same reaction conditions as in the ordinary PCR method. Therefore, the Mg ion concentration is low (1 to
The target gene can be amplified in a reaction system having a high concentration (2 mM) or a conventionally known high concentration (2 to 4 mM).

Prior to the target gene amplification reaction, it is preferable to prepare a standard curve of the gene using a standard gene. For example, a case where the real-time monitoring quantitative PCR method is performed using the above-described fluorescence quenching probe of the present invention as a primer will be described. And
When the amount of decrease in the fluorescence intensity is plotted on a normal graph as a function of the number of cycles, an S-shaped (sigmoid) curve is obtained. The cycle number at the point of the maximum decrease rate and the initial copy number of the target gene (the copy number before the start of PCR), that is, the initial target gene, have an exponential relationship. By preparing a standard straight line of the relationship between the cycle number and the copy number at those times, the initial copy number of the target gene of the unknown sample, that is, the initial target gene amount can be obtained. In addition, quantitative PCR using the above-mentioned fluorescence quenching probe
The method is a new method developed by the present inventors.

The second feature of the quantitative polymorphism analysis method is as follows.
This is an analysis method for analyzing data obtained by the quantitative PCR method. And that is the quantitative PCR described above.
It is the method of analyzing the data obtained by the method itself.
This analysis method is currently the most suitable for measuring the initial target gene amount as accurately as possible.

The present invention also provides a reagent kit used for the above-described quantitative gene amplification method, and a computer-readable recording medium recording a program for causing a computer to execute the above-mentioned data analysis method. . Further, the present invention is a data analysis device having means for performing the above data analysis method.

A third feature of the present invention is a method for analyzing a polymorphism in a gene amplified by the quantitative PCR method of the present invention as described above. Therefore, a polymorphism analysis method will be specifically described. Among various methods for polymorphism analysis, the T-RFLP method can be suitably used in the present invention. Thus, as one example of the present invention, a gene is amplified and the initial gene amount before PCR is measured by a quantitative PCR method using a fluorescence quenching probe as a primer, particularly a real-time monitoring quantitative PCR method. Then, a method of analyzing a polymorphism of the amplified product by the T-RFLP method will be described. The gene amplified using the fluorescence quenching probe as a primer has the 5 ′ end labeled with the fluorescent dye of the present invention.

(1) First, the amplification product is digested with a restriction enzyme. At this time, any of the currently known restriction enzymes can be used, for example, Bso FI, Hha I, Hph
I, Mnl I, Rca I, Alu I, Msp I and the like. Among these, Hha I, A
Hha I is the most preferred, such as lu I and Msp I. The digestion reaction conditions may be the usual conditions for currently known genes. For example, when Hha I is selected as a restriction enzyme, the reaction is carried out at 37 ° C. for 6 hours at a restriction enzyme concentration of 10 units.

(2) The gene fragment digested as described above is preferably denatured by heating to form a single strand. This modification treatment can also be performed under known ordinary conditions. For example, after treatment at 97 ° C. for 5 minutes, the mixture is cooled in ice.

(3) Analysis and Analysis of Gene Fragment In the polymorphism analysis method of the present invention, only a gene fragment labeled with a fluorescent dye is analyzed and analyzed by an electrophoresis method, an HPLC method, a sequencer method, or the like. Become. That is, each band and each peak are detected based on the fluorescence intensity value. Detection can be carried out using conventional analytical instruments currently on the market. For example, ABI 373A (ABI sequencer), ABI
377 (ABI sequencer), Biofocus 3000 (Biorat) and the like.

In the present invention, the appearance of a plurality of bands or a plurality of peaks in the above analysis means that a polymorphism exists. In the case of a single band or a single peak, there is no polymorphism. The ratio of the fluorescence intensity value of each band or each peak is the abundance ratio of the polymorphism. In the above-described quantitative PCR method of the present invention, the amount of the target gene before PCR is measured, so that by multiplying the measured value by the ratio of the polymorphism, the initial abundance of the polymorphism can be obtained. become. With respect to polymorphisms, this method of generating data is only possible with the use of the quantitative PCR method using the fluorescent quenching probe of the present invention.

Further, by containing or attaching a reagent kit for the aforementioned quantitative PCR method,
A convenient kit for quantitative polymorphism analysis. The method for analyzing data of real-time monitoring quantitative PCR may be performed on a computer-readable recording medium on which a program for causing a computer to execute a method of analyzing data obtained by the polymorphism analysis method is recorded. In addition, by storing a program to be executed by a computer, a more convenient recording medium for data analysis by a computer-readable quantitative polymorphism analysis method can be provided. In addition, a polymorphism analyzer having means for a quantitative polymorphism analysis method is provided with a data analyzer for a PCR method, thereby providing a convenient polymorphism analyzer.

[0157]

Next, the present invention will be described more specifically with reference to examples. Examples 1 to 7 relate to the fluorescent probe of the present invention. Example 1 Synthesis of Nucleic Acid Probe : The base sequence of a target nucleic acid is composed of oligodeoxyribonucleotide (5 ′) GGGGGGAAAAAAAAA
As (3 ′), synthesis of the nucleic acid probe of the present invention was performed in the following order.

Design of nucleic acid probe : Since the base sequence of the target nucleic acid is (5 ′) GGGGGGAAAAAAAAA (3 ′), the base sequence of the nucleic acid probe is simply designed as (5 ′) TTTTTTTTTCCCCCC (3 ′) consisting of oligodeoxyribonucleotide. did it. The nucleic acid probe of the present invention was designed as follows.
The fluorescent dye Texas Red (Texas Re
d) is the C-position of the 6th thymine base ring from the 5 'end
The quencher substance Dabcyl was labeled on the OH group of (Texas Red- (5 ') TTTTTT (Dabcyl-) TTTCCCCCC (3') design).

5 'Amino-Modifier C6 kit (Glen Resear
(Ch. USA) was used to modify the phosphate group of thymidyl acid with an amino linker (protecting group MMT). Amino-Modifier C2dT
Using a kit (Glen Research, USA), an OH group at the 6-position C of the base ring of thymidine is converted to an amino linker (protecting group TF).
A). Using these modified thymidylic acid and thymidine, oligonucleotides having the following base sequences were synthesized using a DNA synthesizer (ABI394) (Perkin Elmer Japan Applied). That is, (5 ')
A deoxyribooligonucleotide having a base sequence of TTTTTTTTTCCCCCC (3 ′), wherein the phosphate group at the 5 ′ end is the amino linker (protecting group MMT), and the OH at the 6-position C of the 6th thymine base ring from the 5 ′ end The group is modified with an amino linker (protecting group TFA). The DNA was synthesized by the β-cyanoethyl phosphoramidite method. After synthesis, elimination of the protecting groups was carried out at 55 ° C. and 5% with 28% aqueous ammonia.
Went on time.

Purification of synthesized product : The obtained synthetic oligonucleotide was dried to obtain a dried product. 0.5M Na
It was dissolved in an HCO ₃ / Na ₂ CO ₃ buffer (pH 9.0). The lysate was subjected to gel filtration using a NAP-10 column (manufactured by Pharmacia) to remove unreacted substances.

Labeling of quencher substance : The filtrate was dried and dissolved in 150 μL of sterilized water (oligonucleotide A solution). 1 mg of Dabcyl-NHS (Molecular Probes
Co., USA) with 100 μL of DMF (dimethylformamide)
And the oligonucleotide A solution, 1M Na
150 μL of HCO ₃ / Na ₂ CO ₃ buffer was added, and the mixture was stirred and reacted at room temperature overnight.

Purification of synthesized product : The reaction product was subjected to gel filtration with NAP-25 (manufactured by Pharmacia) to remove unreacted products. Then, the protecting group (MMT) at the 5 'end was removed with 2% TFA. Perform reverse phase HPLC using a SEP-PAC _C18 column,
Linker of the oligonucleotide - (CH _{2) 7} -NH ₂
Was bound to the quencher substance Dabcyl. The fraction was subjected to gel filtration using NAP-10 (Pharmacia).

Labeling of fluorescent dye : The gel filtrate was dried and dissolved in 150 μL of sterilized water (oligonucleotide B solution). 1mg Sulforhodamine 101 Acid Chlori
de (DOJINDO, Japan) was dissolved in 100 μL of DMF, and the oligonucleotide B solution, 1 M NaHCO ₃ / N
a ₂ CO ₃ buffer 150 μL was added, and after stirring, 1
The reaction was performed overnight, and the fluorescent dye Texas Red (Texas-Red) was bound to the amino linker at the 5 'end.

Purification of synthesized product : The reaction product was subjected to gel filtration with NAP-25 (manufactured by Pharmacia) to remove unreacted substances. The present invention in which the same reverse phase HPLC is performed as described above, and the oligonucleotide is obtained by binding the quencher substance to the thymine base at the seventh position from the 5 'end, and the fluorescent dye Texas Red (Texas-Red) is added to the 5' end. , Ie, the nucleic acid probe of the present invention labeled with a fluorescent dye and a quencher substance. The nucleic acid probe of the present invention was eluted later than the oligonucleotide to which the quencher substance was bound.

Quantification of the nucleic acid probe of the present invention was performed by measuring a value at 260 nm with a spectrophotometer. The probe was measured for 650 using a spectrophotometer.
As a result of scanning at an absorbance of nm to 220 nm, it was confirmed that there was absorption of Dabcyl, Texas Red, and DNA. Furthermore, the purity of the purified product was determined by reversed-phase HPLC in the same manner as described above, and it was confirmed that the product was a single peak. As described above, in the synthesized nucleic acid probe of the present invention, at least two complementary bases are present between the base strands where the fluorescent dye Texas Red and the quencher substance Dabcyl are labeled. There is no array.
Therefore, no double chain is formed in the self-chain. That is, no stem loop structure is formed.

The conditions for the above reverse phase chromatography are as follows. And elution solvent _{A: 0.05N TEAA 5% CH 3} CN · eluting Solvent B (for gradient (gradient)):
0.05N TEAA 40% CH ₃ CN ・ Column: SEP-PAK C18; 6 × 250mm ・ Elution rate: 1.0ml / min ・ Temperature: 40 ℃ ・ Detection: 254nm

Example 2 Synthesis of Target Nucleic Acid : An oligonucleotide having the nucleotide sequence of (5 ′) GGGGGGAAAAAAAAA (3 ′) was synthesized in the same manner as in the synthesis of the above-described oligonucleotide, and the target nucleic acid to which the present invention was applicable. And

Example 3 Antibody obtained by hybridizing a probe of the present invention to a target nucleic acid
Responsive fluorescence intensity measurement : quartz cell (10 mm x 10 mm)
(4 mL volume) in 500 μL of buffer (2M NaCl, 20
0 mM Tris-HCl: pH = 7.2), then add
60 μL of sterile distilled water was added and stirred. There, 8.
0 μL of the nucleic acid probe (10 μM) solution of the present invention was added and stirred. The fluorescence intensity was measured over time while keeping the temperature at 35 ° C. (excitation wavelength: 581 nm (8 nm width)), and measured fluorescence wavelength: 603 nm (8 nm width). Then 160
32.0 μL of an nM target nucleic acid solution was added and stirred. Then, the fluorescence intensity was measured over time under the same conditions as described above. The result is shown in FIG. From the figure, it can be seen that the addition of the target nucleic acid increases the fluorescence intensity, and the degree of the increase becomes constant within a very short time, that is, within 100 seconds (1 minute and 40 seconds). Need a minute: Nature Biotechnology, 14, pp. 303-308, 1998). This indicates that the nucleic acid measuring method of the present invention can be performed in a short time.

Example 4 Measurement of Target Nucleic Acid: The fluorescence intensity was measured for each concentration under the same conditions as described above except that the concentration of the target nucleic acid was variously changed.
The result is shown in FIG. From the figure, it was found that the fluorescence intensity also increased according to the concentration of the target nucleic acid, and this relationship was proportional. From the above results, it is recognized that accurate nucleic acid measurement can be performed using the nucleic acid probe of the present invention.

Example 5 Influence of distance between fluorescent dye and quencher substance A deoxyribooligonucleotide having the base sequence shown in FIG. 3 was synthesized in the same manner as in Example 1. In the same manner as in Example 1, as a probe, a fluorescent dye Texas Red was labeled at the phosphate group at the 5 ′ end of the deoxyribooligonucleotide, and a quencher substance Dabcyl was labeled at the OH group at the 6-position C of the thymine base. Then, the labeled thymine base was moved one base at a time to the 3 ′ end. Twenty kinds of such probes of the present invention were synthesized. Each probe was hybridized to a complementary target deoxyribooligonucleotide, and the amount of change in fluorescence intensity before and after hybridization was measured.

500 μl in a quartz cell (same as in Example 3)
Tris buffer (2M NaCl, 200 mM Tri)
s-HCl, pH 7.2) was added. Then 1460μ
One liter of sterile distilled water was added and stirred. There 10μM
Was added and stirred (final concentration of the probe: 40 nM). The fluorescence was measured while keeping the temperature at 35 ° C (excitation: 581 nm, fluorescence: 603 n).
m, slit width: 8 nm (both). Then 10
3 μM target deoxyribooligonucleotide solution
2.0 μl was added and stirred (final concentration of target deoxyribooligonucleotide: 160 nM). Then, the fluorescence measurement was performed over time under the same conditions as described above.

FIG. 4 shows the result. As is apparent from this figure, the fluorescence-emitting probe of the present invention, which has been double-modified with Dabcyl and Texas Red, shows that in most probes, when hybridized with the target deoxyribooligonucleotide, the amount of fluorescence emitted before the hybridization was lower than that before hybridization. , An increase in fluorescence was observed. The maximum fluorescence was observed when the distance between the base having the Texas Red-labeled phosphate group and the base of the Dabcyl-labeled base (when the base number of the base labeled with Texas Red was 0) was 6 bases. Was done. At this time, the amount of emitted fluorescent light was about 11 times. Also, when the distance between bases was 16 bases, a large amount of fluorescent coloring was observed, and the amount of fluorescent coloring at this time was about 11 times as with 6 bases. The helix of DNA is 1 in 10 bases
From the rotation, the 6th and 16th from the 5 'terminal base
The third base is almost on the back of the helix. Therefore, by labeling the 6th and 16th bases with a quencher substance, when the deoxyribooligonucleotide was single-stranded, fluorescence quenching due to electron transfer between Dabcyl and TexasRed occurred. As a result of the separation of Dabcyl and Texas Red, the fluorescence quenching due to electron transfer is released,
exas Red was considered to have developed fluorescence.

Example 6 Relationship Between Fluorescent Dye and Fluorescent Color Amount The type of fluorescent dye in the fluorescent probe of the present invention was examined. The experiment was performed as in Example 5. However, the distance between bases between the fluorescent dye and Dabcyl was 6 bases. Excitation of the slit width for fluorescence measurement and measurement of 5n
m. The results are shown in Table 1. Although the absorption of Dabcyl, which is a quencher, is in the range of 400 to 500 nm, many probes that emit a large amount of fluorescent light emit the fluorescent light on the longer wavelength side than 550 nm, largely deviating from the absorption of Dabcyl. This phenomenon is considered that in the case of a fluorescent dye that emits fluorescence on the longer wavelength side than 550 nm, the fluorescence quenching mechanism of Dabcyl is mainly due to photoexcited electron transfer instead of FRET. Due to the change in the three-dimensional structure of the probe,
Because Dabcyl and the fluorescent dye are physically separated,
The fluorescence quenching phenomenon due to photoexcited electron transfer is canceled. FI
In the case of a fluorescent dye that emits fluorescence having a wavelength close to the absorption of Dabcyl such as TC, the Dab is physically changed due to a change in the three-dimensional structure.
Even if cyl and the fluorescent dye were separated from each other and the fluorescence quenching phenomenon caused by the photoexcited electron transfer was canceled, the fluorescence quenching due to FRET occupied a large part, so it was considered that the fluorescence coloring from the FRET fluorescence quenching did not occur much. Therefore, the fluorescent dye used in the fluorescent probe of the present invention desirably satisfies the following three conditions. That is, (1) it is a fluorescent dye whose fluorescence quenching phenomenon due to photoexcited electron transfer occurs with Dabcyl. (2) It is a fluorescent dye that emits fluorescence at a wavelength far outside the absorption of Dabcyl. (3) It is a fluorescent dye that has a strong interaction with Dabcyl because it suppresses the fluorescence intensity before hybridization (so that the fluorescence quenching phenomenon due to photoexcited electron transfer is more likely to occur).

[0174]

Example 7 Probe Modified in Nucleotide Base with Fluorescent Dye and Quencher As shown in FIG. 5, a probe modified in base in a chain with a fluorescent dye and quencher and a target deoxyribooligonucleotide were used as follows. Except for the above point, it was synthesized in the same manner as in Example 1. (1) 5'Amino-Modifier C6 kit (Glen Res
Amino-Mod instead of using earch, USA)
Texas Red was modified to a probe by a method using ifier C6 dT (manufactured by Glen Research, USA). (2) 5'Amino-M
Instead of modifying the probe with Dabcyl via the odifierC6 kit (Glen Research, USA), Dabcyl d
Dabcyl was directly introduced into the base chain using T (Glen Research, USA). (3) Therefore, the Dabcyl modification step and the subsequent purification step were omitted. Then, whether or not the obtained probe can be actually used was examined in the same manner as in Example 5. In addition, quencher (Dabcyl) and fluorescent dye (Te
xas Red) The effect of the distance between labeled bases was also examined. FIG.
Shows the results. As is clear from these results, it was found that a probe in which a base in a chain was modified with a fluorescent dye or a quencher can actually be used. In addition, the maximum fluorescence was observed when the distance between the bases of Texas Red and Dabcyl was 6 bases or 16 bases, similarly to the case where Texas Red was modified at the phosphate group at the 5 ′ end. At this time, the amount of fluorescent color development was about 10 times that before hybridization.

Examples 8 to 31 and Comparative Example 1 relate to the fluorescence quenching probe of the present invention. Example 8 It hybridizes to the nucleic acid base sequence of 16S rRNA of Escherichia coli , that is, (5 ′) CTGCCTCCCG TAGGAGT
A nucleic acid probe having the nucleotide sequence of (3 ′) was prepared as follows.

Preparation of nucleic acid probe : (5 ') CTGCCTCCCG TAG
An oligodeoxyribonucleotide having a base sequence of GAGT (3 '), in which-(CH ₂ ) ₇ -NH ₂ is bonded to the phosphate group at the 5' end of the oligodeoxyribonucleotide, is available from Medland Certified Residential Co. ( Midland Certified Reag
ent Company, USA). Furthermore, from Molecular Probes, a Fluo Reporter Kit F-6082 (Bodypie FL)
Succinimidyl propionate (BODIPYFL pro
In addition to pionic 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 purchased oligonucleotide to synthesize a nucleic acid probe labeled with bodypi FL used in this example.

[0178]Synthetic purification: Dry the resulting compound to dryness
A solid was obtained. 0.5M NaHCO_Three/ Na _TwoCO_ThreeBuffer solution (pH
9.0). The lysate is applied to a NAP-25 column
Unreacted matter is removed by gel filtration using
Was. Further, reverse phase HPLC (B gradient: 15 to 65%, 25 minutes)
) Was performed under the following conditions. And elute main pipe
The work was separated. Lyophilize the collected fractions and
Nucleic acid in 23% yield from 2 mM oligonucleotide material
A probe was obtained.

The conditions for the above reverse phase chromatography are as follows. And elution solvent _{A: 0.05N TEAA 5% CH 3} CN · eluting Solvent B (for gradient (gradient)):
0.05N TEAA 40% CH ₃ CN ・ Column: CAPCEL PAK C18; 6 × 250mm ・ Elution rate: 1.0ml / min ・ Temperature: 40 ℃ ・ Detection: 254nm

Example 9 Sterilized Nutrient Broth (NB) (manufactured by Difco)
Escherichia coli JM109 strain was cultured overnight at 37 ° C. in a 200 ml (sterilized) Erlenmeyer flask to which 50 ml of liquid medium (composition: NB, 0.08 g / 100 ml) was added. 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 cells. The cells were washed once with 100 μl of 30 mM phosphate buffer (soda salt) (pH: 7.2). The cells were treated with the phosphate buffer 100 containing 130 mM NaCl.
Suspended in μl. The suspension is 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 prepare a homogenate.

After centrifuging the homogenate, 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. 3
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 fluorometer.

The emission amount of the fluorescent dye before hybridization was measured using a 30 mM phosphate buffer (soda salt) (pH: 7.2) containing 130 mM NaCl instead of the supernatant. The value was adopted. The amount of luminescence was measured by changing the ratio of the amount of the nucleic acid probe to the amount of the supernatant (excitation light 503n).
m; measured fluorescence color 512 nm). The result is shown in FIG.
As can be seen from FIG. 7, the emission of the fluorescent dye decreased as the ratio of the supernatant increased. That is, in the present invention, it can be seen that the amount of 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 10 Preparation of Nucleic Acid Probe: The oligonucleotide having the nucleotide sequence of (5 ′) CCCACATCGTTTTGTCTGGG (3 ′) hybridizes to 23S rRNA of Escherichia coli JM109 strain. ,-(CH ₂ ) ₇ -NH ₂ was combined with the oligonucleotide of Midland Certified Resin Co., Ltd. in the same manner as in Example 8.
fied Reagent Company, USA). Furthermore, as in Example 8, a molecular probe (Molecular Prob
es) from the Fluoro Reporter Kit (FluoReporter K)
it) F-6082 (BODIPY FL propionic acid succinimidyl
ester) and a kit containing a reagent that binds the compound to an amine derivative of an oligonucleotide). 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 8 to obtain a nucleic acid probe labeled with Bodepy FL in a yield of 25% from the initial oligonucleotide starting material of 2 mM.

Example 11 Pseudomonas paucimobilis 421Y strain (E. coli JM109 strain) obtained in Example 9 was prepared using the same medium and culture conditions as in Example 9.
(Current name: Sphingomonas pusubimovils) (FERM
P-5122) was mixed at the same concentration with E. coli JM109 strain at OD660 value to prepare a composite microorganism system. The resulting mixture (E. coli
A homogenate was prepared in the same manner as in Example 9 for the bacterial cell concentration of the JM109 strain). Example 1
The same experiment as in Example 9 was carried out using the nucleic acid probe prepared in Example 0, and the same result as in Example 9 was obtained.

Example 12 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 (30mer) shown below (pol
ya-poly j) was prepared using a DNA synthesizer ABI394 (Perkin Elmer, USA).

Further, a bodepy FL is added to the 5 ′ end of the deoxyribooligonucleotide corresponding to the above-mentioned synthetic deoxyribooligonucleotide (target gene or target nucleic acid).
The following probe of the present invention labeled with was prepared. 5 ′ of deoxyribooligonucleotide which is a primer corresponding to the above synthetic deoxyribooligonucleotide
A primer in which-(CH ₂ ) ₆ -NH ₂ is bonded to the terminal phosphate group is used as a primer at Medland Certified Reagent Company (Midland Certified Reagent Company, USA)
Purchased from. In addition, molecular probes (Molecula
r Probes) from Fluo Reporter Kit (FluoRepo)
rter Kit) F-6082 (BODIPY FL propionic acid succinidy of FL / C6)
kit containing a reagent for binding the compound to an amine derivative of deoxyribooligonucleotide) in addition to the ester). The kit is reacted with the deoxyribooligonucleotide of the purchased primer, and the probe of the present invention labeled with the following bodypy FL:
d and fh were synthesized. Then, the degree of decrease in the emission of the fluorescent dye (that is, the degree of quenching) 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. The purification was performed in the same manner as in Example 8.

Name: Target deoxyribooligonucleotide ・ poly a 5′ATATATATTTTTTTTGTTTTTTTTTTTTTT3 ′ ・ poly b 5′ATATATATTTTTTTTTGTTTTTTTTTTTTTT3 ′ ・ poly c 5′ATATATATTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Name: target deoxyribooligonucleotide ・ poly f 5'ATATATATTTTTTTTCTTTTTTTTTTTTTT3 '・ poly g 5'ATATATATTTTTTTTTCTTTTTTTTTTTTT3' ・ poly h 5'ATATATATTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Name: Probe of the present invention probe a 3'TATATATAAAAAAAACAA5'-BODIPY FL / C6 probe b 3'TATATATAAAAAAAAACA5'-BODIPY FL / C6 probe c 3'TATATATAAAAAAAAAAC5'-BODIPY FL / C6 probe d 3 ' TATATATAAAAAAAAAAA5'-BODIPY FL / C6

Name: Probe of the present invention probe f 3'TATATATAAAAAAAAGAA5'-BODIPY FL / C6 probe g 3'TATATATAAAAAAAAAGA5'-BODIPY FL / C6 probe h 3'TATATATAAAAAAAAAAG5'-BODIPY FL / C6

(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: 503 nm • Measurement fluorescence color: 512 nm

[0192]

The results are shown in Table 2. As can be seen from Table 2, the nucleic acid probe labeled with a fluorescent dye
A (oligodeoxyribonucleotide), when the probe is hybridized to the target DNA at the terminal, the terminal base is 1 to 3
It is preferable that the base sequence of the probe is designed so that G (guanine) is present in the base sequence of the target DNA at least one base or more apart from the base. In addition, when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target DNA, the probe is designed so that a plurality of base pairs of the nucleic acid hybrid complex form at least one pair of G and C at the terminal portion. It can be seen from Table 2 that the base sequence is preferably designed.

Example 13 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.

Name: target deoxyribooligonucleotide poly k 5'TATATATATATTTTTGGGGG3 'poly l 5'TATATATATATTTTTTGGGG3' poly m 5'TATATATATATTTTTTTTTGGG3 'poly n 5'TATATATATATTTTTTTTGG3' poly o 5'TATATATATATTTTTTTTTG3

Name: Target deoxyribooligonucleotide ・ poly p 5'TATATATATATTTTTCCCCC3 '・ poly q 5'TATATATATATTTTTTCCCC3' ・ poly r 5'TATATATATATTTTTTTCCC3 '・ poly s 5'TATATATATATTTTTTTTCC3' ・ poly t 5'TATATATTTTTTTTTTTTTTTTTTTTTTTT '

Name: Probe of the present invention probe k 3'ATATATATATAAAAACCCCC5'-BODIPY FL / C6 probe l 3'ATATATATATAAAAAAAACCCC5'-BODIPY FL / C6 probe m 3'ATATATATATAAAAAAACCC5'-BODIPY FL / C6 probe 3 ' ATATATATATAAAAAAAACC5'-BODIPY FL / C6 ・ probe o 3'ATATATATATAAAAAAAAAC5'-BODIPY FL / C6

Name: Probe of the present invention probe p 3'ATATATATATAAAAAGGGGG5'-BODIPY FL / C6 probe q 3'ATATATATATAAAAAAGGGG5'-BODIPY FL / C6 prober 3'ATATATATATAAAAAAAGGG5'-BODIPY FL / C6'probes ATATATATATAAAAAAAAGG5'-BODIPY FL / C6 ・ probe t 3'ATATATATATAAAAAAAAAG5'-BODIPY FL / C6 ・ probe u 3'ATATATATATAAAAAAAAAA5'-BODIPY FL / C6

[0199]

As can be seen from Table 3, 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 fluorescence intensity.

Example 14 A target nucleic acid having the following nucleotide sequence and a nucleic acid probe of the present invention were prepared in the same manner as described above. In addition, the nucleic acid probe of the present invention in the present example is obtained by labeling the 5 ′ end of the oligonucleotide with bodypy FL / C6. 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 Examples.

Name: Target deoxyribooligonucleotide poly W 5'CCCCCCTTTTTTTTTTTT 3 'poly X 5'GGGGGGAAAAAAAAAAAA 3' poly Y 5'TTTTTTCCCCCCCCCCCC 3 'poly Z 5'AAAAAAGGGGGGGGGGGG 3'

Name: Probe of the present invention probe w BODIPY FL / C6-5'AAAAAAAAAGGGGGG 3 'probe x BODIPY FL / C6-5'TTTTTTTTTCCCCCC 3' probe y BODIPY FL / C6-5'GGGGGGGGGAAAAAA 3 'probe z BODIPY FL / C6-5'CCCCCCCCCTTTTTT 3 '

[0204]

As can be seen from Table 4 and the above embodiment,
(I) When the terminus of the probe of the present invention labeled with a fluorescent dye is composed of C and the target nucleic acid hybridizes, G
When forming the C pair, (ii) when the end of the probe of the present invention labeled with a fluorescent dye is composed of a base other than C, the base at the position labeled with the fluorescent dye and the base of the target nucleic acid G at the 3 'end of the target nucleic acid from the base pair
When at least one is present, the decrease rate of the fluorescence intensity is large.

Example 15 Preparation of Probe and Target Nucleic Acid (Target) for Fluorescence Quenching Probe with Intrachain Modification Nucleic acid probes a and b of the present invention having the following base sequences:
In addition, target nucleic acids c and d of oligodeoxyribonucleotide were prepared by the following method. After introducing an amino linker into the probe sequence using Amio-Modifier C6 dC (manufactured by Grin Research) in the oligodeoxyribonucleotide chain of the nucleotide sequence shown in probes a and b, the amino linker was post-labeled with Bodipy FL. Was used as the probe of the present invention (the underlined portion is the Amino-Modifier C6 dC introduction site). The other synthesis methods are the same as in Example 8. As shown below, probe a is modified at one site in the deoxyribooligonucleotide chain and probe b is modified at five sites by Bodipy FL. The synthesis of the oligodeoxyribonucleotide, the method for modifying the oligodeoxyribonucleotide with Bodipy FL, the method for purifying the probe, and the like are the same as those described in the above Examples.

Probe a: 5'TTT C (-Bodipy FL) TTTTTT CCCCCCCCC3 'Probe b: 5'TTT C (-Bodipy FL) TT C (-Bodipy FL) TT C (-
Bodipy FL) CC C (-Bodipy FL) CC C (-Bodipy FL) CCC 3 'Target nucleic acid c: 5'GGGGGGGGAA AAAAGAAA3' (target of probe a) Target nucleic acid d: 5'GGGGGGGGGA AGAAGAAA 3 '(probe b
Target)

Measurement Method The concentration of the added probe was 8 μM (final concentration), and the concentration of the added target nucleic acid was 32 μM (final concentration). The fluorescence excitation wavelength was 499 nm, and the fluorescence measurement wavelength was 522 nm.
The slit width was 5 nm each. Conditions other than the above were performed by the same experimental method as the base selectivity of the target nucleic acid in the fluorescence quenching phenomenon of Example 12.

Experimental Results From the following Table 5, it was revealed that when the probe a or b hybridized to the target nucleic acid, the fluorescence intensity was significantly reduced (fluorescence quenched) before and after the hybridization. Therefore, even when one or several fluorescent dyes are modified to C in the same DNA probe chain, one fluorescent dye is replaced with a 5 ′ terminal phosphate group of the oligonucleotide, or a 3 ′ terminal ribose or deoxyribose. When the OH group at the 3'-position is modified (in this case, the base at the 5'-end or 3'-end of the nucleic acid probe forms a GC pair with the target nucleic acid. --- Japanese Patent Application No. 2001-1)
33529), the fluorescence intensity was found to decrease. Also, it can be seen that the initial fluorescence intensity value of probe b is about four times higher than the initial fluorescence intensity value of probe a. Therefore, it was suggested that by labeling C in the same chain with a plurality of fluorescent dyes, the detection sensitivity was significantly improved without impairing the property of decreasing the fluorescence intensity. It is presumed that the decrease in the fluorescence intensity is caused by whether the emission energy of the fluorescent dye is transferred to G or another substance due to the interaction with G. This phenomenon was discovered for the first time in the world by the present inventors.

Example 16 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 was the probe z of Example 14, and the target nucleic acid was the oligonucleotide z of Example 14. The results are shown in Table 6. As can be seen from the table, the preferred fluorescent dyes for use in the present invention are FITC, BODIPY F
L, BODIPY FL / C3, BODIPY FL / C6, 6-joe, TMR and the like.

[0211]

Example 17 Heating of Target Nucleic Acid (16S rRNA)
Experiment of effect of treatment) Preparation of nucleic acid probe of the present invention: 16S rRNA of Escherichia coli JM109 strain
KYM-7 corresponding to the nucleotide sequence from bases 1156 to 1190
Specifically hybridizes to the 16S rRNA nucleotide sequence of the strain
(5 ') CATCCCCACC TTCCT CCGAG TTGACCCCGG CAGTC (3')
(35 base pairs) having a base sequence of 1 to 16 and 25 to
35th base is deoxyribonucleotide, 17-24 salt
Modification of the OH group at the 2'-position carbon with a methyl group (ether
Modified with a bond).
The OH group of the phosphate group at the 5'-terminal of-(CH _Two)₇-NN_TwoQualify with
The oligonucleotide bound by the
Dorand Certified Refining Company
(Midland Certified Reagent Company, USA)
Purchased. In addition, a 2-o-Me probe (2-o-Me oligonucleotide)
The probe consisting of otide is simply called 2-o-Me probe.
U. The 2-o-Me oligonucleotide used for) is GEN
Outsourced synthesis to SET Corporation (France) and obtained
It is. Further, the molecular probe is used in the same manner as in Example 8.
Fluor Reporter Kit from (Molecular Probes)
(FluoReporter Kit) F-6082 (Bodypie FL / C6
Succinimidyl onate (BODIPY FL / C6 propio
nic acid succinimidyl ester)
Including reagents that bind to amine derivatives of oligonucleotides
Kit). The kit is
A book labeled with Bodepy FL / C6 that acts on nucleotides
A nucleic acid probe of the invention was synthesized. Implement the resulting composite
Purified as in Example 8 to obtain the first oligonucleotide material
The present invention labeled with bodypi FL / C6 in a yield of 23% from 2 mM
A light nucleic acid probe was obtained. This probe is used for 35 base chain 2-
It was named O-Me probe.

5 ′) An oligoribonucleotide having the nucleotide sequence of TCCTTTGAGT TCCCGGCCGG A (3 ′) was synthesized using a DNA synthesizer in the same manner as described above, and
Type helper probe. On the other hand, (5 ') CCCTGGTCGT A
An oligoribonucleotide having the nucleotide sequence of AGGGCCATG ATGACTTGAC GT (3 ') was synthesized using a DNA synthesizer in the same manner as described above to obtain a backward type, that is, a reverse type helper probe.

A 35-base chain 2-O-Me probe, a forward type helper probe and a reverse type helper probe were each dissolved in a buffer solution having the following composition so as to have a concentration of 25 nM, and heated to 75 ° C. (probe solution) ). 16Sr
After heat-treating the RNA at 95 ° C. for 5 minutes, it was added to the above-mentioned probe solution under the following reaction conditions, and the fluorescence intensity was measured with a fluorescence measuring instrument Perkin Elmer LS-50B. The result is shown in FIG. The control using 16S rRNA not subjected to the heat treatment was used as a control. It can be seen from FIG. 8 that the decrease in the fluorescence intensity is large in the heat-treated experimental plot. The result is 16Sr
Heat treatment of RNA at 95 ° C. shows stronger hybridization with the probe of the present invention. Reaction conditions: 16S rRNA: 10.0 nM Probe: 25 nM each Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulfite, pH
5.1 Temperature: 75 ° C

Example 18 (Experiment of Contribution of 2'-O-Me Oligonucleotide and Helper Probe to Improvement of Hybridization Efficiency) The following various nucleic acid probes of the present invention and various helper probes that hybridize to the 16S rRNA described above. Was prepared in the same manner as in Example 17 above. In addition, all 2-O-Me oligonucleotides used for 2-o-Me probes are GENSET
It was obtained by commissioning a synthesis company (France). Then, under the following conditions, the effects of the 2-o-Me probe of the present invention, the effect of the length of the base chain of the probe, and the effect of the helper probe were examined by the experiments in FIGS. 9A, 9B, 9C, and 9D below. The system was examined in the same manner as in Example 17.
The result is shown in FIG. The figure shows that the 2-o-Me probe of the present invention contributes to the hybridization efficiency. Moreover, when the base chain of the 2-o-Me probe is short, the helper probe is useful for increasing the hybridization efficiency.

1) 35-base chain 2-o-Me probe: the same probe as in Example 17, 2) 35-base chain DNA probe: 35-base chain of 1) above
A probe having the same base sequence as the 2-o-Me probe, but having an oligonucleotide composed of deoxyribose; 3) a 17-base chain 2-O-Me probe: the 35-base chain of the above 1)
A probe having the same base sequence as the 2-O-Me probe, but having 8 bases removed from the 5 'end and 10 bases removed from the 3' end. 4) 17 base strand DNA probe: 33 bases of the above 2) A probe having the same base sequence as a strand DNA probe, but having 16 nucleotides removed from the 3 ′ end,

5) Forward type 2-o-Me helper probe: the 2'-position carbon of ribose at the central 8 bases (9 to 16 bases counted from the 5 'end) of the forward type helper probe of Example 17 Helper probe in which the OH group of (1) is modified (ether bond) with a methyl group. 6) Reverse type 2-O-Me helper probe: Example 1 described above.
7 reverse-type helper probes at the central 8 bases (5 ′
2 'of ribose (9 to 16 bases counted from the end)
7) a forward type DNA helper probe: the same base sequence as that of the forward type helper probe of Example 17, except that the oligonucleotide is Helper probe composed of deoxyribonucleotide; 8) reverse-type DNA helper probe: Example 1 above
7) A helper probe having the same nucleotide sequence as that of the reverse type helper probe, but the oligonucleotide is composed of deoxyribonucleotides. 9) 35 nucleotide oligoribonucleotides: (5 ′) CATCCCCACC
TTCCTCCGAG TTGA CCCCGG Oligoribonucleotide having a base sequence of CAGTC (3 ′), 10) 17-base chain oligoribonucleotide: (5 ′) CCTTCC
An oligoribonucleotide having a nucleotide sequence of TCCG AGTTGAC (3 ′).

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 sulfite
pH 5.1 Reaction temperature: 35 base chain 2-O-Me probe: 75 ° C. 17 base chain 2-O-Me probe: 70 ° C. 35 base chain DNA probe: 75 ° C. 17 base chain oligoribonucleotide DNA probe :
60 ° C

Experimental system, FIG. 9A: HP (M) +: 16S rRNA, 35 base strand DNA probe, forward 2-O-Me helper probe,
Reverse 2-O-Me helper probe, HP (D) +: 16S rRNA, 35 base strand DNA probe, forward DNA helper probe, reverse DNA helper probe, HP-: 16S rRNA, 35 base strand DNA probe, Ref . (Control): 35 base strand DNA oligoribonucleotide, 35 base strand

Experimental system, FIG. 9B: HP (M) +: 16S rRNA, 35 base strand 2-O-
Me probe, forward type 2-O-Me helper probe, reverse type 2-O-Me helper probe, HP (D) +: 16S rRNA, 35 base chain 2-O-
Me probe, forward DNA helper probe, reverse type DNA helper probe, HP-: 16S rRNA, 35 base chain 2-O-Me probe, Ref. (Control): 35 base chain DNA oligoribonucleotide, 35 base chain 2-O-Me probe.

Experimental system, FIG. 9C: HP + (M): 16S rRNA, 17 base strand DNA probe, forward type 2-O-Me helper probe,
Reverse type 2-O-Me helper probe, HP + (D): 16S rRNA, 17 base strand DNA probe, forward type DNA helper probe, reverse type DNA helper probe, HP-: 16S rRNA, 17 base strand DNA probe, Ref . (Control): 17-base strand DNA oligoribonucleotide, 17-base strand

Experimental system, FIG. 9D: HP + (M): 16S rRNA, 17 base strand 2-O-
Me probe, forward type 2-O-Me helper probe, reverse type 2-O-Me helper probe, HP + (D): 16S rRNA, 17 base chain 2-O-
Me probe, forward type DNA helper probe,
Reverse type DNA helper probe, HP-: 16S rRNA, 17 base chain 2-O-Me probe, Ref. (Control): 17-base strand DNA oligoribonucleotide, 17-base strand 2-O-Me probe.

Example 19 (Preparation of Calibration Curve for rRNA Measurement) The rRNA was heated at 95 ° C. for 5 minutes at various concentrations ranging from 0.1 to 10 nM. Added to the reaction solution under the reaction conditions,
After 0 seconds, the decrease in fluorescence intensity was measured using a Perkin Elmer LS-50B. The results are shown in FIG. The figure shows that the calibration curve shows linearity at 0.1 to 10 nM. The following 35 base chain 2-o-Me probe was used in Example 17
Is the same probe as. Reaction conditions: 35 base chain 2-O-Me probe: 1.0 to 25 nM Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulfite,
pH 5.1-Reaction temperature: 75 ° C

Example 20 (FISH method) Cellulomonas sp.KYM-7 (FERM P-11339)
And Agrobacterium sp.KYM-8 (FE
The following 35- or 36-base oligodeoxyribonucleotide 2-o-Me probe of the present invention which hybridizes to each rRNA of RMP-16806) was prepared in the same manner as described above. The base sequence of each probe is as follows.

KYM-7 Cellulomonas sp. KYM-7 35-Base Oligodeoxyribonucleotide 2-o-
Me probe: (5 ') CATCCCCACC TTCCTC CGAG TTGA CCCCGG
CAGTC (3 ') (The underlined portion is modified with a methyl group.) 36-base oligodeoxyribonucleotide 2-o-Me probe for measuring rRNA of Agrobacterium sp. KYM-8: (5 ') CATCCCCACC TTCCTC TCGG CTTAT CACCG GCAGT
C (3 ') (The underlined portion is modified with a methyl group.)

[0226] Cellulomonas sp. KYM-7 and Agrobacterium sp. KYM-8 were mixed-cultured in a medium having the following medium composition under the following culture conditions, and the culture was collected every culture time. From them, rRNA was converted to RNeasy Maxikit (QIAGEN
Was prepared by using After heating the rRNA at 95 ° C. for 5 minutes, it was added to a reaction solution that had been previously set under reaction conditions.
After reacting for 1000 seconds, the fluorescence intensity was measured using Perkin Elmer LS-50B. The result is shown in FIG.
It was shown to. The total rRNA was RiboGree
n) total RNA Quantification Kit (Company name: molecular probes) Location: Eugen
e, Oregon, USA). As can be seen from the figure, 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.

[0227] - 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 · 7H
₂ O, 0.2; NaCl, 0.1; (NH ₄ ) ₂ SO ₄ ; 0.1. • 100 ml of the medium 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 cultured in advance on a slant medium. One platinum loop of cells was taken from the slant medium and inoculated into the sterilized conical flask medium. 30 ° C, 150 rpm
m.

Reaction conditions: 35 base chain oligodeoxyribonucleotide 2-o-
Me probe: 1.0 to 10 nM Buffer: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5% lithium dodecyl sulfite,
pH 5.1-Reaction temperature: 75 ° C

Example 21 (Example of fluorescence quenching probe with intrachain modification) A target nucleic acid having the following base sequence and a nucleic acid probe of the present invention were prepared. To provide probes a), b),
After introducing an amino linker into the corresponding base sequence using Amino-Modifier C6 dC (manufactured by Glen Research),
This amino linker was labeled BODIPY FL. Other synthesis methods are the same as in Example 8. Therefore the probe a)
Is fluorescently modified not at the phosphate group at the 5 ′ end but at the C base at the 5 ′ end. BODIPY FL modification method, purification method, etc.
Same as above. Probe a) 5 'C (-BODIPY FL) TTTTTTTTTCCCCCCCCC 3' Probe b) 5 'TTTC (-BODIPY FL) TTTTTTCCCCCCCCC 3' Probe nucleic acid of probe a) C) 5 'GGGGGGGGGAAAAAAAAG 3' Probe nucleic acid of probe b) d) 5 'GGGGGGGGGAAAAAGAAA 3'

<Experimental Method> An experiment was conducted in the same manner as in Example 9. <Experimental results> As is clear from the table below, it was revealed that the fluorescence intensity decreases when both the probe a) and the probe b) hybridize to the target nucleic acid. From the results of probe b), it was clarified that fluorescence modification of cytosine bases in the DNA strand other than the 5 ′ end or 3 ′ end also functions as a fluorescence quenching probe.
It is also clear from the results of probe a) that even for cytosine at the terminal, a fluorescent quenching probe can be obtained by fluorescently modifying a site other than the phosphate group at the 5′-terminal or the OH group at the 3′-terminal. became.

[0231]

Example 22 describes a method for analyzing or measuring a polymorphism and mutation of a target nucleic acid. Example 22 Four kinds of oligonucleotides having the base sequences shown below were synthesized using the DNA synthesizer of Example 12 described above.
In the same manner as in Example 12, 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 could be evaluated from the change in fluorescence intensity. The nucleotide sequence of the nucleic acid probe of the present invention has G at any 3 ′ end of the target oligonucleotide.
Is designed to match 100% to the nucleotide sequence of the oligonucleotide when it exists. The hybridization temperature was set at 40 ° C. at which 100% of the base-pairs between 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 12.

The probe of the present invention: 3'TTTTTTTTGGGGGGGG
C5'BODIPY FL / C6-Target oligonucleotide No. 1: 5'AAAAAAAACCCCCC
CCA3 ′ • Target oligonucleotide No. 2: 5'AAAAAAAACCCCCC
CCC3 '-Target oligonucleotide No. 3: 5'AAAAAAAACCCCCC
CCI3 '(I: hypoxanthine)-Target oligonucleotide No. 4: 5'AAAAAAAACCCCCC
CCG3 '

Table 8 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.

[0235]

In the present invention, target nucleic acids (for example, data obtained by a method for analyzing or measuring polymorphisms and / or mutations of the above target oligonucleotide Nos. 1 to 4 (for example, data of columns A and B in Table 8)) In the analyzing method, the fluorescence intensity value of the reaction system when the target nucleic acid is hybridized with the nucleic acid probe of the present invention (the nucleic acid probe described above) is corrected by the fluorescence intensity value of the reaction system when the target nucleic acid is not hybridized. The arithmetic processing means the calculation of (AB) / A in Table 4. 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 23 FIG. 12 shows an example of a DNA chip model of the present invention. First, a modified probe prepared by introducing an amino group into the OH group at the 3'-position carbon of the 3'-terminal ribose of 3'TTTTTTTTGGGGGGGGC5'BODIPY FL / C6 which is the probe of the present invention prepared in Example 22, and A surface-treated slide glass was prepared by treating the surface of the slide glass with a silane coupling agent having an epoxy group as a reactive group. The solution containing the modified probe was spotted on the surface-treated slide glass using a DNA chip preparation device GMS ^™ 417ARRAYER (TAKARA). Then, the modified probe bound to the glass surface at the 3 'end. The slide glass was placed in a closed container for about 4 hours to complete the reaction. Then, the slide glass was alternately soaked twice in a 0.2% SDS solution and water for about one minute. Further, a boron solution (water 300)
1.0 g of NaBH ₄ dissolved in ml. ) For about 5 minutes. 9
After immersing in water at 5 ° C. for 2 minutes, the reagent was quickly immersed alternately twice in a 0.2% SDS solution and water for about 1 minute to wash away the reagent. Dry at room temperature. Thus, the DNA chip of the present invention was prepared.

Further, by providing a minute temperature sensor and a heater as shown in the figure at positions corresponding to each spot of the modified probe on the lower surface of the glass, high performance can be imparted to the DNA chip of the present invention. A case where a target nucleic acid is measured using this DNA chip will be described. When the target nucleic acid is not hybridized to the probe, or when the probe does not form a GC pair at the fluorescent dye-labeled end even when hybridized, or 1 to 3 bases from the terminal base portion where the probe and the target nucleic acid are hybridized Away,
When at least one G (guanine) or cytosine (C) does not exist in the base sequence of the target nucleic acid, the fluorescence intensity does not change. However, conversely, when a target nucleic acid is hybridized to the probe, or when a GC pair is formed at the fluorescent dye-labeled end even when hybridized, or when the probe and the target nucleic acid are hybridized. When at least one G (guanine) or cytosine (C) exists in the base sequence of the target nucleic acid at a distance of one to three bases from the terminal base, the fluorescence intensity decreases. This fluorescence intensity was measured using a DNA chip analyzer GMS ^™ 418 Array Scanner (TAKA).
RA).

Example 24: Detection experiment of single nucleotide polymorphism (SNPs) using the DNA chip of the present invention I) Preparation of target nucleic acid: (5 ') AAACGATGTG GGAAGGCCCA GACA
G CCAGG ATGTTGGCTT AGAAGCAGCC (3 ') oligodeoxyribonucleotide having the nucleotide sequence of a DNA synthesizer ABI394
(Perkin Elmer, USA) to obtain a target nucleic acid. II) Preparation of nucleic acid probe: 15 from 5 ′ end of target nucleic acid
The following six oligodeoxyribonucleotides having a base sequence that hybridizes to the base sequence (underlined part) were synthesized using a DNA synthesizer ABI394 (Perkin Elmer,
(USA). And 3'-Amino-Modifier
C7 CPG (Glen Research, Catalog No. 20-295
Using 3), the 3'-OH group of the 3'-terminal deoxyribose was aminated. Further, the phosphate group at the 5 'end was labeled with BODIPYFL in the same manner as in Example 12.

1) Probe 100 (100% match):
(5 ') CCTTCCCACA TCGTTT (3'), 2) Probe-T (1 base mismatch): (5 ') CCTTCCCA
TA TCGTTT (3 '), 3) Probe-A (1 base mismatch): (5') CCTTCCCA
AA TCGTTT (3 '), 4) Probe-G (1 base mismatch): (5') CCTTCCCA
GA TCGTTT (3 '), 5) Probe-TG (2 base mismatch): (5') CCTTCC
CTGA TCGTTT (3 '), 6) Probe-TGT (3 base mismatch): (5') CCTT
CCCTGT TCGTTT (3 '),

III) Preparation of DNA chip All DNA probes were prepared using 0.1 M MES (2-Morpholinoethan).
esulfonic acid) buffer solution (pH: 6.5), 500nM
It was a solution of the concentration. DNA microarrayer (D
This is a manual chip arrayer consisting of NA microarrayer No. 439702, 32-pin type, and DNA slide index No. 439701. Slide glass (Black silylated) for DNA chip using Greiner
The probe solution was spotted on slides (manufactured by Greiner). After the completion of the spot, the DNA probe was reacted with the slide glass at room temperature for 60 minutes in a wet chamber, and the probe was immobilized on the slide glass. Thereafter, the plate was washed with 50 mM TE buffer (pH: 7.2). In addition, four spots were spotted for each probe solution. After immobilization, slide the glass with 0.1% SDS
(sodium dodecylsulfate) solution, washed twice with distilled water, and washed with sodium borate solution (2.5 mg-
NaBH ₄ / ml-25% ethanol solution) for 5 minutes.
Then, it was immersed in a hot water bath heated to 95 ° C. for 3 minutes and dried. FIG. 12 shows a schematic diagram of the DNA chip of the present invention. When the probe of the present invention immobilized on a slide glass does not hybridize to the target nucleic acid, BODIPY F
L is colored, but when hybridized, the color is less than when not hybridized,
That is, it decreases. The slide glass is heated by a micro heater (in the present invention, the slide glass was heated using a transparent heating plate for a microscope (MP-10MH-PG, Kitasato Supply Co., Ltd.) as shown below).

IV) Detection and measurement of SNPs 100 μM target nucleic acid solutionμ50 mM TE buffer (p
H: 7.2) The｝ was placed on the DNA chip prepared as described above. It was covered with a cover glass, and the cover glass was sealed with nail polish so that the target nucleic acid did not leak. FIG. 13 shows an outline of devices for detection and measurement.
First, a transparent heating plate for a microscope (MP-10MH-PG, Kitasato Supply Co., Ltd.) was placed on a sample stage of an Olympus erect focus laser microscope (AX80 type). The DNA chip of the present invention prepared above was placed on the plate, the temperature of the plate was changed from 95 ° C. to 33 ° C. in steps of 3 ° C., and the target nucleic acid and the probe were reacted for 30 minutes. The fluorescence intensity of the reaction process of each spot was measured in an image capture format using a cooled CCD camera (C4880-40, Hamamatsu Photonics). The captured image is converted to an image analyzer (image analysis software (TPlab spectrum; S
(Ignal Analytics, Verginia) was installed on a personal computer (NEC)), the brightness of each spot was calculated, and the relationship between temperature and brightness was obtained.

The results of the experiment are shown in FIG. From the figure, it can be seen that the fluorescence intensity is reduced for all the probes. Therefore, the dissociation curve between the probe of the present invention and the target nucleic acid can be easily monitored by the method of the present invention.
In addition, since the difference between the Tm value of the probe 100 that matches 100% with the target nucleic acid and the probe that mismatches by one base is 10 ° C. or more, both can be easily identified from the dissociation curve. That is, it is understood that SNPs can be easily analyzed by using the DNA chip of the present invention.

The PCR of the present invention is described in Examples 25 to 28 below.
The method is described. Example 25 Using 16S rRNA gene in Escherichia coli genomic DNA as a target nucleic acid, (BODIPY FL /
A primer (a nucleic acid probe of the present invention) labeled with C6) was prepared.

Preparation of Primer 1 (Eu800R: reverse type): An oligodeoxyribonucleotide having the base sequence of (5 ′) CATCGTTTAC GGCGTGGAC (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 the bound oligonucleotide _{2) 9} -NH _2, were purchased from the Midland-Sateifaido-Rejindo Company, Inc. (USA). Furthermore,
Fluo Reporter Kits F-6082 (Succinimidyl propionate of BODIPY FL / C6 (BODIPY FL propio)
In addition to nic acid succinimidyl ester), a kit containing a reagent for binding the compound to an amine derivative of an oligonucleotide was purchased. 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 the 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. Furthermore, reverse phase HPLC (B gradient: 15-65%, 25
Min) under the following conditions. Then, the eluting main peak was collected. The fractionated fraction was freeze-dried to give the primer 1 of the present invention 2 m of the first oligonucleotide material.
M obtained in 50% yield.

The conditions for the above reverse phase chromatography are as follows. And elution solvent _{A: 0.05N TEAA 5% CH 3} CN · eluting Solvent B (for gradient (gradient)):
0.05N TEAA 40% CH ₃ CN ・ Column: CAPCEL PAK C18; 6 × 250mm ・ Elution rate: 1.0ml / min ・ Temperature: 40 ℃ ・ Detection: 254nm

Example 26 Preparation of Primer 2 (Eu500R / forward: forward type): Fluorescent dye (BODIPY FL / C6) at the 5 ′ end of oligodeoxyribonucleotide having the base sequence of (5 ′) CCAGCAGCCG CGGTAATAC (3 ′) Primer 2 labeled with
It was prepared in the same manner as in a yield of 50%.

Example 27 Using a test tube containing 5 ml of sterilized nutrient broth (NB) (manufactured by Difco) liquid medium (composition: NB, 0.08 g / 100 ml), E. coli JM109 strain was incubated at 37 ° C. overnight. 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 this cell, DNeasy Tissue Kit
Genome D using (QIAGEN, Germany)
NA was extracted. The extraction followed the protocol of this kit. As a result, a 17 ng / μl DNA solution was obtained.

Example 28 Using the above-described Escherichia coli genomic DNA, 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 by replacing the nucleic acid probe (two probes utilizing the FRET phenomenon) and a normal primer (a normal primer not labeled with a fluorescent dye) according to the present invention with the present invention. Primer 1
And / or 2 was performed according to the procedure manual.

The PCR was performed in a hybridization solution of the following components. E. coli genomic DNA solution: 3.5 μl (final concentration 0-6)
(ng / 20 μl) (final copy number: 0 to 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 ・ all Volume: 20.0 μl In addition, Escherichia coli 16S rDNA as a target nucleic acid was used at the concentration of the experimental section shown in the explanatory column of FIG. Experiments were performed with / or 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 were obtained from DN of 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 diluted 10-fold and used. Also, Taq Start is sold by Clonetech (USA)
An antibody for Taq DNA polymerase, which can be added to the reaction solution to suppress the activity of Taq DNA polymerase up to 70 ° C. That is, the hot start (ho
t start).

The reaction conditions are as follows. The measurement was performed using a LightCycler ^™ system (manufactured by Roche Diagnostics). At that time, among the F1 to F3 detectors in the system, the F1 detector was used, and the gain of the detector was fixed at 10 and the excitation intensity was fixed at 75.

FIGS. 15 and 16 show the results. FIG.
5 and 16, 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. In the figure, the amount of decrease in the emission of the fluorescent dye is expressed as the decrease in the fluorescence intensity. FIG. 17 shows E. coli 16SrD expressing the copy number of E. coli 16S rDNA as a function of cycle number.
2 shows a calibration curve of NA. 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 concentration of the target nucleic acid can be measured.

Example 29 In Example 28, PCR was performed using the probe of the present invention as a primer. In this example, the probe of the present invention was replaced with the probe of the present invention instead of the two probes utilizing the FRET phenomenon. 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: (5 ') AGAGTTTGAT CCTGGCT
CAG (3 ') ・ Reverse primer E1492R: (5') GGTTACCTTG TTACGA
CTT (3 ') c) Probe used: BODIPY FL- (5') CGGGCGGTGT GTAC
(3 ′) (However, the 3 ′ end is phosphorylated) d) PCR measuring instrument used: LightCycler ^™ system e) PCR conditions: Denaturation reaction: 95 ° C., 10 seconds (first time only, 60
Annealing reaction: 50 ° C, 5 seconds Nucleic acid extension reaction: 72 ° C, 70 seconds Total number of cycles: 70 cycles

F) Fluorescence measurement (measured once 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 (for each nucleotide)

FIG. 18 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.

The following examples describe the data analysis method of the present invention for analyzing data obtained by using the above-described quantitative PCR method of the present invention. Example 30 Human genomic DNA (human β-globin)
(TaKaRa catalog product number 9060) (TaK
Using aRa Co., Ltd. (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 ') CTGGTCTCCT TAAACCTGTC An oligodeoxyribonucleotide having a base sequence of TTG (3') was converted to a DNA synthesizer ABI3.
94 (manufactured by Perkin Elmer, USA), and the phosphate group at the 5 'end of the oligodeoxyribonucleotide was treated with phosphatase to give cytosine. The 5'-carbon OH group of cytosine was replaced with-( CH ₂ ) ₉ -NH ₂ was purchased from Midland Certified Raised Company. Furthermore, a reporter kit (FluoReporter Kit) F-6082 (succinimidyl propionate of bodepy FL / C6 (B
In addition to ODIPY FL propionic acid succinimidyl esters), a kit containing a reagent for binding the compound to an amine derivative of an oligonucleotide) was purchased. Acting the kit on the purchased oligonucleotide,
Primer KM38 labeled with bodypy FL / C6 of the present invention
+ C was synthesized.

Purification of the 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: 15-65%, 25 minutes)
Was performed under the following conditions. Then, the eluting main peak was collected. The collected fraction was freeze-dried to obtain the primer KM38 + C of the present invention in a yield of 50% from the initial oligonucleotide starting material of 2 mM.

The conditions for the above-mentioned reverse phase chromatography are as follows. And elution solvent _{A: 0.05N TEAA 5% CH 3} CN · eluting Solvent B (for gradient (gradient)):
0.05N TEAA 40% CH ₃ CN ・ Column: CAPCEL PAK C18; 6 × 250mm ・ Elution rate: 1.0ml / min ・ Temperature: 40 ℃ ・ Detection: 254nm

Example 31 Preparation of primer KM29 (forward type): (5 ′) GGTT
An oligodeoxyribonucleotide having the base sequence of GGCCAA TCTACTCCCA GG (3 ′) was synthesized in the same manner as in Example 27.

Comparative Example 1 This comparative example is for data analysis which does not have an arithmetic processing step (the processing of the formula (1)) in which the fluorescence intensity value during the nucleic acid extension reaction is divided by the fluorescence intensity value during the thermal denaturation reaction. It concerns the use of software. Using the above-mentioned human genomic DNA, primers KM38 + C and primer KM29, a PCR reaction was carried out using a LightCycler ^™ system, and the fluorescence intensity in each cycle was measured. The PCR of this comparative example uses the primer labeled with the fluorescent dye described above, and is a novel real-time quantitative PCR method for measuring a decrease, not an increase, in fluorescence emission. Data analysis was performed using the software of the system. In the PCR of this comparative example, the primer of the present invention is used 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. KM38
Except using + C and KM29, it carried out according to the procedure manual of the said apparatus.

The PCR was performed in a hybridization solution of the following components. Human genomic DNA: 1.0 μl (final concentration 1 to 1000
・ 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 FIG. The experiment was carried out at the concentrations in the experimental plots indicated in the detailed description columns. The final concentration of MgCl ₂ was 2 mM.

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

The Taq solution and Taq DNA polymerase solution are commercially available from Roche Diagnostics, Inc.
NA Master Hybridization Probe (DNA Ma
sterHybridization Probes) kit. In particular
Taq DNA polymerase solution is 10 × conc. (Red cap) was diluted 10-fold and used. In addition, Taq Start was launched by T
This is an antibody for aq 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-Re-denaturation reaction (from the second time on): 95 ° C, 10 seconds-Annealing reaction: 60 ° C, 5 seconds-DNA extension reaction: 72 ° C, 17 seconds The measurement was performed using a light cycler. ^This was performed using a ^TM system.
At this time, of 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 PCR was performed as described above, and the fluorescence intensity in each cycle was measured. The result is shown in FIG. 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 any cycle, the fluorescence intensity value is constant during the denaturation reaction, but during the nucleic acid extension reaction, it is observed that the fluorescence intensity decreases from around the 25th cycle. So the reduction is human genomic DNA
It can be seen that this occurs in the order of increasing copy numbers.

As shown in FIG. 19, the fluorescence intensity value at the initial cycle number was not uniform for each copy number of human genomic DNA. Therefore, the following steps (b) to (j) were added to the data analysis method used in this comparative 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 the fluorescence intensity value in each cycle, F _n (72) = the fluorescence intensity value at 72 ° C. in each cycle,
F ₁₀ (72) = the fluorescence intensity value after the extension reaction at 72 ° C. at the 10th cycle. (C) displaying and / or printing each converted value obtained in the step (b) on a display as a function of the number of cycles;

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

(F) The data processed in the process of (d) is used as a threshold value of 0.1.
5, a step of counting the number of cycles reaching the value, and (g) creating a graph in which the value counted 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; (i) calculating the correlation coefficient or the relational expression of the straight line drawn in the step (h). (J) displaying and / or printing the correlation coefficient or the relational expression calculated in the step (i) on a display.

Using the data analysis software described above, the data obtained in FIG. 19 was processed as follows following the above. FIG. 20 shows the data processed in the above process (b) printed (process (c)). That is, the fluorescence intensity of each cycle is converted with the fluorescence intensity value of the 10th cycle as 1, and the converted value is plotted against the corresponding cycle number. FIG.
Is a printout of the data processed in step (d) (step (e)). That is, the reduction rate (quenching rate) of the fluorescence intensity is calculated from each plot value in FIG. 20, and each calculated value is plotted with respect to each cycle number.

FIG. 22 is a graph in which the data prepared in the step (f) is printed with the graph created in the step (g) (the step (h)). That is, the fluorescence intensity decrease rate = 0.5 is set to a threshold (thresh).
Old), 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 (R
2) was calculated in the step (i) and printed (the step (j)), and was 0.9514. Thus, it was impossible to obtain an accurate copy number with this correlation coefficient.

Example 32 (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. Data processing is
The same process as in Comparative Example 1 was performed except that the following process (a) was performed before the process (b) in Comparative Experimental Example 1, and the processes (b) and (d) were changed as follows. Was. (A) The fluorescence intensity value of the reaction system when the amplified nucleic acid in each cycle is hybridized with the nucleic acid probe of the present invention (labeled with a fluorescent dye) (that is, at the time of nucleic acid extension reaction (72 ° C.) Of the reaction system measured when the nucleic acid hybrid complex (the amplified nucleic acid hybridized with the nucleic acid primer) is dissociated (that is, at the completion of the nucleic acid heat denaturation reaction (9)).
5)), the correction calculation process divided by the fluorescence intensity value),
The measured fluorescence intensity value was corrected by the following [Equation 1]. f _n = f _{hyb, n} / f _{den, n} [Formula 1] [where, f _n = correction value of fluorescence intensity in each cycle, f _{hyb, n}
= Fluorescence intensity value at 72 ° C. in each cycle, f _{den, n} = Fluorescence intensity value at 95 ° C. in each cycle] FIG. 23 shows the obtained values plotted against the number of cycles.

(B) A process of calculating the fluorescence change rate (decrease rate or extinction rate) between each sample in each cycle by substituting the correction calculation processing value in [Equation 1] for each cycle into [Equation 3]. 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) The arithmetic processing value of each cycle obtained in the process of (b) is applied to an arithmetic processing for obtaining a logarithmic value of a change rate (decrease rate or extinction rate) of the fluorescence intensity according to [Equation 6]. Affixing process, that is, a process of performing an arithmetic process by the following [Expression 11], log ₁₀ {(1-F _n ) × 100} [Expression 11] [where, F _n = value obtained by [Expression 10]] . [Equation 1
1] is a case where b = 10 and A = 100 in [Equation 6].

The above results are shown in FIGS. FIG. 24 is a graph in which the values processed in the processes (a) and (b) are plotted against the number of cycles and printed.
FIG. 25 is a graph in which the values obtained by processing the values obtained in FIG. 24 as in the process (d) are plotted against the number of cycles and printed.

Next, based on the graph of FIG. 25, processing was carried out in the steps (f), (g) and (h). That is,
Based on the graph in FIG. 25, 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. The result is shown in FIG. The correlation coefficients (R ² ) obtained by processing these calibration curves in the processes (i) and (j) 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 33 (Melting curve analysis of nucleic acid and Tm
Example of value analysis) For nucleic acids amplified by the novel PCR method of the present invention, 1) a process of gradually increasing or decreasing the temperature from a low temperature until the nucleic acid is completely denatured (for example, from 50 ° C to 95 ° C) 2) a step of measuring the fluorescence intensity at short time intervals (for example, an interval corresponding to a temperature rise of 0.2 ° C. to 0.5 ° C.) in the step 1); Displaying on the display as a function of time, ie displaying the melting curve of the nucleic acid; 4) first differentiating the melting curve of step 3);
Create software consisting of displaying the differential value of the process (-dF / dT, F: fluorescence intensity, T: time) on the display; 6) obtaining the inflection point from the differential value obtained in the above 5) Then, it was combined with the data analysis software of the present invention. 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 30 was performed on 1 and 10 copies of the same human genomic DNA as in Example 32.
27, and the data processed in the steps 1), 2), 3), 4) and 5) are printed as shown in FIG. FIG. 28 is a diagram of a nucleic acid melting curve obtained by treating 1 and 10 copies of the 75th amplification product in the steps 1), 2) and 3) of this example. FIG. 29 shows the result of differentiating this curve in the process 4) and obtaining the inflection point (Tm value) in the processes 5) and 6). From FIG. 29, it was found that each amplification product was a different product because the Tm values of the amplification products of 1 copy and 10 copies were different.

The following example is an example of a quantitative polymorphism analysis method. Example 34 Fluorescence Quenching Probe of the Present Invention: Preparation of Probes Eu47F and Eu1392R (1) Synthesis of Fluorescence Quenching Probe Eu47F A fluorescent quenching probe Eu47F labeled with a bodypy FL as described below was attached to the terminal phosphate group as described below.
(PerkinElmer, USA).

(2) Synthesis of Eu1392R A deoxyribooligonucleotide having the base sequence of (5 ′) TTGTACACACCGCCCGTCA (3 ′) was synthesized.

The above deoxyribooligonucleotides
-(CH_Two)₆-NH _TwoDeoki combined with
Use Ciribooligonucleotides as Medland
Midland Certified R
eagent Company, USA). In addition,
Fluoro Report from Molecular Probes
Tar Kit (FluoReporter Kits) F-6082 (Bodypie FL
Succinidimyl propionate (BODIPY FL pr
opionic acid succinidimyl esters)
To bind the product to the amine derivative of the oligonucleotide
Kit containing the drug). Purchase the kit
To act on the deoxyribooligonucleotide
The above-described fluorescent quenching probe of the present invention labeled with bodypy FL
Was synthesized.

The purification of the synthesized product was carried out as follows. The synthesized product was dried to obtain a dried product. 0.5M Na ₂ CO ₃ /
It was dissolved in NaHCO ₃ buffer (pH 9.0). Transfer the lysate to NAP-2
Gel filtration was performed with 5 columns (manufactured by Pharmacia) to remove unreacted substances. Furthermore, reverse phase HPLC (B gradient: 15 to 65
%, 25 minutes) under the following conditions. Then, the eluting main peak was collected. The fractions collected were freeze-dried to obtain the target compound at a yield of 50% from 2 mM of the initial oligonucleotide raw material.

Conditions for reverse phase chromatography: 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 35 (1) Culture of Escherichia coli JM109 Strain 53 medium (composition: casein peptone (trypsin digest of casein), 10 g; yeast extract, 5 g; glucose, 5 g; salt, 5 g; distilled water, 1000 mL) The Escherichia coli JM109 strain was then cultured (medium 50 mL / 250 mL conical flask, shaking culture at 37 ° C. for 12 hours).
Then, cells were collected from the culture solution (centrifugation 10,000,
Washed twice with distilled water at 0 rpm for 5 minutes).

(2) Preparation of 16S rRNA cDNA Total RNA was extracted from the cells using a SOGEN kit (Nippon Gene) according to the protocol of this kit. Then, BcaBEST ^™ RNA PCR kit (Takara Shuzo Co., Ltd.)
The extract was subjected to amplification and reverse transcription reaction (RT-PCR) for 16 sRNA under known conditions in accordance with the protocol of this kit. At that time, the aforementioned fluorescence quenching probe Eu1392R of the present invention was used as a primer. Subsequently, RNA was degraded by Rnase H (3
(0 ° C., 20 minutes), a pure cDNA of the 16S rRNA gene was obtained. cDNA concentration was measured using the OliGreen ^R ssDNA Quantitation kit (Molecular Probes).

Example 36 (1) Quantitative PCR, Data Analysis, and Preparation of Calibration Curve for cDNA The above cDNA solution was treated with the fluorescence quenching probe Eu47F of the present invention as a forward primer and Eu139.
Real-time monitoring quantitative PCR reaction was performed using 2R as reverse plummer. LightCycler ^TM Syst as a real-time monitoring quantitative PCR device
The reaction was carried out using em (Roche Diagnostics, Germany) according to the procedure described in the procedure manual. In addition, TaKaRaTaq ^™ (Takara Shuzo Co., Ltd.) was used as a DNA polymerase.

The PCR was performed with the following components. E. coli cDNA: 1.0 μl (final concentration 10 ² to 10 ⁶
・ Primer solution: 4.0 μl (final concentration: 0.1 μM) ・TaKaRaTaq ^™ : 10.0 μl (0.5 Units) ・ Milli-Q pure water: 5.0 μl ・ Total volume: 20.0 μl The experiment was performed using the copy number of the experimental section indicated in the note 30. Final concentration of MgCl ₂ is 2 mM
Met.

The reaction conditions were as follows. The measurement conditions were as follows.・ Excitation light: 488 nm ・ Measured fluorescent color: 530 nm

Under the above conditions, real-time monitoring quantitative PCR was performed, and the fluorescence intensity of each cycle was actually measured. The measured values were analyzed according to the data analysis method of the present invention. That is, the data was processed in the following process. (A) Fluorescence intensity value of the reaction system when the amplified nucleic acid in each cycle is hybridized with a nucleic acid primer labeled with a fluorescent dye (ie, at the end of nucleic acid extension reaction (72 ° C.)
Of the reaction system when the amplified nucleic acid hybridized with the nucleic acid primer is completely dissociated (ie, the fluorescence intensity value at the end of the nucleic acid thermal denaturation reaction (96 ° C.)). The correction calculation process, that is, the actually measured fluorescence intensity value was corrected by [Equation 1]. f _n = f _{hyb, n} / f _{den, n} [Formula 1] [where, f _n = correction value of the fluorescence intensity of the cycle, f _{hyb, n} =
Fluorescence intensity value at 72 ° C. for each cycle, f _{den, n} = fluorescence intensity value at 96 ° C. for each cycle]

(B) The correction processing value in [Equation 1] in each cycle is substituted into [Equation 3], and the calculation process of calculating the fluorescence extinction ratio between samples in each cycle, ie, the following [Equation 3] course of processing at 10], F _n = [in equation 10] [wherein processing values of F _n = each cycle, f _n = [equation 1] f _n / f ₂₅ values for each cycle obtained in, f ₂₅ = the value obtained by [Equation 1] and the number of cycles is 25]. [Formula 1
0] is obtained when a = 25 in [Equation 3].

(C) 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, the 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]]. [Formula 1
1] is obtained when b = 10 and A = 100 in [Equation 6].

FIG. 30 shows the above result. FIG.
The values calculated in steps (a), (b) and (c) are
It is plotted against the number of cycles and printed.

Next, based on the graph of FIG. 30, the following (d)
And in the process of (e). (D) Among the data processed in the step (c),
The process of thresholding 0.2 and calculating the number of cycles that has reached that value. (E) A step of preparing a graph in which the values calculated in the step (d) are plotted on the X-axis and the copy number before the start of the reaction is plotted on the Y-axis, that is, a calibration curve of E. coli cDNA (FIG. 31). FIG.
Uses the data obtained by the quantitative PCR method of the present invention as a data analysis method of the present invention, that is, (a), (b),
It is the final result processed in the process of (c), (d), and (e). FIG. 31 shows 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 37 (1) Construction of polymorphism system (complex microbial system) The 10 bacterial strains shown in Table 6 were isolated from DSMZ (Deutsche Samm
Lung von Mikroorganismen und Zellkulturen GmbH) and each strain was separately cultured using the 53 medium described above. The culturing conditions are the same as those for Escherichia coli. The cells were collected from each culture (centrifugation was performed at 10,0 cells).
00 rpm, 10 minutes, washed twice with distilled water). Total RNA was extracted from each of the cells using a SOGEN kit (Nippon Gene) in the same manner as described above.

[0298] 1: Paracoccus pantotrophus, 2: Sphingomonas natator
ia, 3: Bdellovibriostolpii, 4: Microbacterium impe
riale, 5: Pseudomonas fluorescens, 6: Agromyces me
dislanum, 7: Cellulomonas cellulans, 8: Brevibacte
rium liquefaciens, 9: Leminorella grimontii, 10: Rh
odococcus luteus

Thereafter, the pure cS of the 16S rRNA gene of each strain was
DNA was obtained. The cDNA concentration of each of the obtained 10 strains was measured in the same manner as in the case of Escherichia coli described above. For a solution with a known cDNA concentration, 300,000 with distilled water
It was diluted to copy / μL. For 10 strains,
A mixture obtained by mixing the diluents in equivalent amounts was defined as a composite microorganism system, that is, a polymorph system (hereinafter, referred to as a polymorph system). This polymorphism includes
CDNA of 10 strains is 300,000 copy /
Since it is contained at a concentration of μL, a total of 3,000
This means that cDNA was contained at a concentration of 0000 copy / μL.

(2) Real-time monitoring quantitative P
CR The cDNA of the polymorphism system was subjected to real-time monitoring quantitative PCR in the same manner as in the case of Escherichia coli, using the fluorescence quenching probes Eu47F and Eu1392R of the present invention as a common pramer. A polymorphic sample was prepared at an absolute concentration of 300,000 copy / 20 μL (20 μL of the entire reaction solution).
L) was added to the reaction solution. In the real-time monitoring quantitative PCR of the polymorphism system, a decrease in the fluorescence intensity was observed, and the reaction was stopped at 22 cycles, which is the exponential amplification phase of the gene (see FIG. 30). When the threshold was set to log Rn (fluorescence quenching rate) = 0.2, the copy number of the cDNA of the reaction solution of the real-time monitoring quantitative PCR performed on the polymorphic system was 288,000 copies (FIG. 31). Since the initial addition amount or theoretical value is 300,000 copies,
It was confirmed that the calibration curve prepared by the method of the present invention exhibited good quantitative properties.

Example 38 Polymorphism Analysis (1) Analysis by T-RFLP After performing the PCR reaction as described above, the amplified product was subjected to column (MicroconPCR, Millipore Corporation, Bedfor
d, MA, USA). Purify the product with restriction enzyme H
ha1 (recognition site: GCG / C, / = cut site)
N (overnight) treatment. After completion of the treatment, only the cut fragments are applied to the column (Microcon and Micropure-EZ, Millipore Corporati
on, Bedford, MA, USA). Table 9 shows the size of the cDNA fragment of each strain after restriction enzyme treatment.

The cDNA solution subjected to the column purification was heat-denatured, and then subjected to a sequencer (ABI PRIS
T-RFLP analysis was performed using MTH 310, PE Applied Biosystems). The peak pattern is shown in FIG. Each peak was quantified using a standard BODIPY FL modified fragment of known concentration. As a result of calculating the molar composition of each peak, all the molar compositions were within the range of 9.4 to 10.8, and no extreme difference in the PCR amplification efficiency of the cDNA fragment of each strain was observed ( See Table 9). The copy number of all cDNAs determined by quantitative PCR was multiplied by the molar composition ratio to determine the initial cDNA copy number of each strain (see Table 9). The copy number / initial added copy determined by quantification was 0.89 to 1.04 (see Table 8).
Therefore, the initial cDNA of each strain
It was found that the copy number could be accurately determined.

Example 39 A fluorescent probe was used as a primer (hereinafter, referred to as a fluorescent primer).
Example of real-time quantitative PCR method and quantitative polymorphism analysis method applying this method Real-time quantitative PCR using fluorescent primers
An example of the method and a quantitative polymorphism analysis method using the real-time quantitative PCR method will be described.

1) Experimental Method and Conditions <Preparation of Artificial Complex Microorganism System (Template)> An artificial complex microbial system was prepared, and the effectiveness of the quantitative polymorphism analysis method was proved using this as a model system. For the experiment, ten microorganisms shown in Tabel 9 were purchased from DSMZ. Each strain was separately cultured using 53 medium. The cells are collected from the culture solution, and the kit reagent ISOGEN (Nippon Gene,
Japan) and Total DNA was extracted according to the protocol.
Then Eu47F (CITAACACATGCAAGTCG, I = inosine), Eu13
92R (TTGTACACACCGCCCGTCA) as a primer, 16sRNA
A PCR reaction was performed using the gene as an amplification target. PicoGreen ^R dsDNA Q
After quantification with uantitation Kit (Molecular Probes),
Each was diluted to 300,000 copies / ml with sterile distilled water. A mixture of these in equal amounts was used as a model artificial complex microorganism system. In this model artificial complex microbial system, 1
The amplification products of 16 S rRNA gene of 0 microorganisms were 30,00 respectively.
Contained at a concentration of 0 copies / ml, total 16S rRNA
The gene amplification product concentration will be 300,000 copies / ml.

<Real-Time Quantitative PCR Experiment Using the Fluorescent Primers of the Present Invention> The above-mentioned artificial complex microorganism system (16S rRNA gene mixed sample) was
Quantitative PCR using xas red and Dabcyl doubly modified fluorescent emission primers was performed. Eu47F-mo as common primer
di (CITAACACATGCAAGTCG, I = inosine), Eu1392R (TTGTACA
CACCGCCCGTCA). Eu47F-modi has the same nucleotide sequence as Eu47F, but the ninth T from the 5 'end is Texas.
red, the ninth T is Dabcyl-modified. The modification method of Texas red and Dabcyl was the same as in Example 7. As a quantitative PCR device, iCycler (Bio-Rad (BIO
-RAD). Initial Denature is 60 at 95 ° C
Second, and the PCR cycle is Denature = 95 ° C./60
Seconds, annealing = 50 ° C / 60 seconds, extension = 72 ° C /
The test was performed under the condition of 70 seconds. The PCR reaction was stopped at an exponential amplification region so that the initial gene composition ratio did not collapse (so that no PCR bias was applied). Primer
The concentration was set to 0.1 μM for each of Eu47F and Eu1392R as the final concentration. DNA polymerase is TaKaRa TaqTM (Takara Shuzo) 0.5
Used at a concentration of Units / 20 μl. Mg ion concentration is 2mM
And dNTPs were added to a final concentration of 2.5 mM each. Hot start was performed using an AntiTaq body (manufactured by Clonetech) in accordance with the manufacturer's instructions. A 16s rDNA gene amplification product of E. coli was used as a standard sample for preparing a calibration curve. Preparation of the amplified product of 16 s rDNA gene of E. coli was performed in the same manner as in the artificial complex microorganism system. After preparing the calibration curve, the artificial complex microorganism system was quantified.
Genetic amount of artificial complex microbial system is 300,000 copies in absolute amount
/ 20 μl (20 μl = total amount). The fluorescence measurement was performed once after denaturation and after annealing in each cycle. The fluorescence emission rate was obtained by correcting the fluorescence intensity after annealing (at the time of hybridization) with the fluorescence intensity after denature (at the time of dissociation), similarly to the fluorescence quenching rate.

A specific calculation formula is: Fn = ((f hyb, n / f den, n) / (f hyb, n '/ f den,
n ')} X100 Fluorescence luminescence rate at Fn = n cycle f hyb, Fluorescence intensity at annealing (hybridization) at n = n cycle f den, Fluorescence intensity at denature (dissociation) at n = n cycle f hyb, n ′ = fluorescence intensity during annealing (hybridization) in the cycle (n ′ cycle) before the fluorescence emission from the amplification product occurs f den, n ′ = cycle before the fluorescence emission from the amplification product occurs (n ′ cycle) ) Fluorescence intensity at the time of denature (dissociation)

<Analysis by T-RFLP> After the real-time quantitative PCR reaction was completed, the amplification product was purified by a column (Microcon
PCR, Millipore Corporation, Bedford, MA, USA) and over with Hha1 (recognition site: GCG / C, / = cut site)
Restriction enzyme treatment was performed at night reaction. After heat denaturation of the solution containing the restriction enzyme fragment, T-RFLP analysis was performed using a sequencer (ABI PRISMTH310, PE Applied Biosystems). Thereafter, each restriction enzyme fragment was quantified using a fluorescent probe having the same chain length as a standard substance, and the molar composition ratio of each peak was determined.

2) Results <Real-time quantitative PC using fluorescent primers
R results> The results are shown in FIGS. As can be seen from FIG. 33, it was shown that the amplification product could be monitored using the fluorescent primer. In addition, threshold (thresh hold) value (log Fn (fluorescence rate)
= 1.6) and the number of cycles of the DNA added initially (FIG. 34).
It was shown to. As can be seen from this figure, the cycle number and the copy number of the initial addition have a linear relationship. Therefore, this figure suggests that the initial target gene can be accurately quantified from the number of cycles reaching the threshold value. In the artificial complex microorganism system, the PCR reaction was stopped at the number of cycles (23 cycles) at which logarithmic amplification was observed (see FIG. 33). From the calibration curve shown in FIG. 34,
The 16S rRNA copy number of the artificial complex microbial system is about 296,000 co
quantified as pies. The initial addition amount was 300,000 copies, confirming good quantitative properties of this method.

<Analysis results by T-RFLP> The amplification products of real-time quantitative PCR were analyzed by the T-RFLP method, and the restriction enzyme fragments of the 16S rRNA gene of each strain were quantified. Is 9.5
Ｐ10.6, and P by 16S rRNA genotype
No difference in CR amplification efficiency was observed (see Table 10). The molar copy ratio was multiplied by the total copy number of the 16s rRNA gene determined by quantitative PCR to determine the copy number of the initial 16s rRNA gene of each of the constituent microorganisms (see Table 10). Regarding the 16S rRNA gene of each strain, the copy number / initial added copy number determined by quantification was 0.94 to 1.
05 (see Table 10), it was proved that the quantification of the initial copy number (quantification of the target nucleic acid) of the mixed gene of the artificial complex microbial system of the present method was accurate.

[0310]

Example 40 Experimental Example of Real-Time Quantitative PCR Using Fluorescent Probes Quantitative PCR is performed using primers of the prior art and the fluorescent probe of the present invention, and amplification products are monitored in real time by the probes. An example of the real-time quantitative PCR method based on this principle will be described.

1) Experimental method and conditions <Preparation of template DNA> Paracoccus denitrifica
ns DSM 413 genomic DNA using the DNeasy ^™ Tissue Kit (QIA
After extraction using GEN GmbH, Hilden, Germany, E10F (AG
AGTTTGATCCTGGCTCAG: without fluorescence modification), E1400R (GGTTACC
TTGTTACGACTT) primer set and normal PC
At R, the 16S rRNA gene was amplified. PCR amplification products
Pico GreendsDNA Quantitation Kit (Molecular Probes
Inc.), and then quantified the 16S rRNA gene.
A solution containing 6 ng / μl was prepared.

<Other conditions> The base sequence of the fluorescent probe was 5'CTAATCCTTT- (Texas red) GGCGAT- (Dabcyl) AA
ATC 3 ', which was obtained by modifying the 9th T from the 5' end with Texas red and modifying the 15th T from the 5 'end with DABCYL. The modification method is the same as in Example 7. In addition, the 3 ′ end of the probe was phosphorylated so as to inhibit extension from the 3 ′ end. The same forward and reverse primers as those used in ordinary PCR were used (E10F, E1400R) (that is, primers not modified with a fluorescent dye). Real-time PCR device is iCycler
(Bio-Rad) was used.

[0314] As for the PCR conditions, the first denature was 95% for both ordinary PCR and real-time quantitative PCR.
℃, 120sec, denature is 95 ℃, 60sec, anneal is 56 ℃, 6
0 sec and extension are the conditions of 72 ° C and 70 sec. The Mg ion concentration was 2 mM. dNTPs were added to a final concentration of 2.5 mM each. Gene Taq as Taq polymerase
(Nippon Gene) was used. Primers are used for normal PCR
Both the method and the real-time quantitative PCR method were added at a final concentration of 100 nM. This DNA solution was used as a standard template solution, and was added at a concentration of 0.6 pg to 6 ng / reaction. As a template, Para adjusted by the above method
A 16SrRNA gene amplification product derived from coccus denitrificans DSM413 was used and added to the reaction system at 0.6 pg to 6 ng / reaction. The fluorescent primer was added at 50 nM. The fluorescence measurement was performed once after denaturation and after annealing in each cycle. The fluorescence emission rate was determined in the same manner as in Example 39.

2) Results FIG. 35 shows the results of real-time monitoring of amplification products by the fluorescent probe. From this figure, it was found that it was possible to monitor the amplification product using a fluorescent probe. FIG. 35 shows the relationship between the number of cycles required to reach the threshold value (log Fn (fluorescence rate) = 1.8) and the initial amount of added DNA. As can be seen from this figure, it was found that the cycle number and the copy number of the initial addition had a linear relationship. The correlation coefficient at this time was R2 = 0.9993. Therefore, from this figure, from the number of cycles that reached the threshold value,
It was found that the quantification of the initial copy of the target gene could be performed accurately. From the above results, it was proved that the initial target nucleic acid concentration (the amount of the target nucleic acid existing before amplification) can be measured by the real-time quantitative PCR method using a fluorescent probe.

Example 41 Detection of Single Nucleotide Polymorphism Using a Fluorescent Probe or a Quenching Probe Using a fluorescent probe or a quenching probe,
A method for detecting a single nucleotide polymorphism from a dissociation curve will be described with reference to specific examples.

1) Experimental method As the fluorescent probe, the same fluorescent probe as that used in Example 40 was used. The fluorescent quenching probe has the same nucleotide sequence as the fluorescent luminescent probe, and has a BODIPY F
Those modified with L were used. {(BODIPY FL) -5 'CTAATCC
TTTGGCGATAAATC3 '}. The modification method is the same as in Example 8. The target is a base sequence ((5 ′) GATTTATCGCCA) that is 100% complementary to the above fluorescent probe and the fluorescent quenching probe.
AAGGATTAG (3 ')) and a complementary nucleotide sequence, but a nucleotide sequence containing a single nucleotide polymorphism in which the 10th C from the 5' end has been substituted with T ((5 ') GATTTATCGTCAAAGGATTAG (3')) Was used. The probe was added at a final concentration of 100 nM. Synthetic target DNA was added at a final concentration of 400 nM. The composition of the hybridization solution was the same as that used in Example 12. As the synthetic target DNA, either one of the two types of prepared targets was used. In the experiment, a solution prepared in advance under the above-mentioned conditions was added to a tube for fluorescence measurement, and the temperature was raised from 30 ° C. to 80 ° C. at 0.1 ° C./sec.

From the results of the fluorescence measurement, a dissociation curve between the probe and the target was prepared, and it was evaluated whether a sequence containing a single nucleotide polymorphism could be identified from the difference in the dissociation curves. As an experimental device, iCycler (Bio-Rad) was used. For the fluorescence filter, the fluorescence filter for Texas red provided by Bio-Rad is used for the fluorescence detection of the fluorescent probe.
For fluorescence detection of the fluorescence quenching probe, a fluorescence filter for FITC, also provided by Bio-Rad Inc., was used.

2) Results The results are shown in FIG. From this figure, it was found that the Tm value of the dissociation curve with the target containing the single nucleotide polymorphism was about 10 ° C. lower than the Tm value of the dissociation curve with the 100% complementary target for both the fluorescent probe and the fluorescence quenching probe.
This indicated that the presence or absence of hydrogen bonding for one base appeared as a difference in Tm. From the above, it has been proved that single nucleotide polymorphism can be easily distinguished by using a fluorescence emitting probe or a fluorescence quenching probe.

Example 42 D using a fluorescent probe
NA Chip A DNA chip using a fluorescent probe will be specifically described with reference to Examples.

1) Experimental method A fluorescent probe having the nucleotide sequence shown in Table 11 was prepared.
These are all partial nucleotide sequences of the human CYP21 gene, and include a SNPs site in the probe nucleotide sequence.
The probe name is the Whitehead Institute (http: // wal
The ID number of SNPs of do.wi.mit.edu/cvar_snps/) was used as it is. The synthesis method was the same as in Example 7 except for the following two points. Is the same as (1) At the 5 'end, 5'-Amino-Modif
Amino linker was introduced using ier C12 (Glen Research). (2) Texas red not only has Amino-Modifier C6 dT but also Amino-Modifier C
Modification was also performed using 6 dC (Glen Research). The probe sequences and the modification positions in Texas red and Dabcyl probes are as shown in Table 10. Table 12
The ones shown in Table 1 were used.

[0322]

[0323]

<Preparation of DNA chip>
One spot was performed for each probe solution. The other method of preparing a DNA chip is the same as the method of preparing a DNA chip using the above-described fluorescence quenching probe. The probe of the present invention immobilized on a slide glass, when red does not hybridize to the target nucleic acid, the fluorescence of Texas red is quenched, but when hybridized, the emission of fluorescence is not hybridized. Increase significantly.

<Detection and measurement method of SNPs>
5 kinds of 100% match target mixed solution containing M concentration ｛5
Using 0 mM TE buffer (pH: 7.2) was placed on the DNA chip prepared as described above. Five kinds of 1 mismatch target mixed solutions each containing 100 μM concentration were prepared in the same manner and mounted on a DNA chip different from the one on which the 100% match target mixed solution was mounted. These were covered with a cover glass, and the cover glass was sealed with nail polish so that the target nucleic acid did not leak. Therefore, in this test, a total of two DNA chips were prepared. For each of these chips, perform fluorescence observation continuously while changing the temperature,
A dissociation curve with the target was created.

<Measurement Apparatus> The equipment for detection and measurement is the same as that shown in FIG.

2) Experimental Results The experimental results are shown in FIG. From the figure, it can be seen that the fluorescence intensities of all the probes increase as the temperature decreases. This indicates that each fluorescent probe hybridized with the corresponding target base sequence.
Therefore, it was shown that the dissociation curve between the probe of the present invention and the target nucleic acid can be easily monitored by the method of the present invention. In addition, since the difference between the Tm value of the probe that matched 100% with the target nucleic acid and the probe that mismatched by one base was around 10 ° C. in the present study, both could be easily identified from the dissociation curve. That is, D of the present invention
This experiment showed that multiple types of SNPs can be analyzed simultaneously by using an NA chip.

Example 43 Gene amplification on a DNA chip on which a fluorescent probe and a fluorescent quenching probe were immobilized and real-time detection of the amplified product DN with a fluorescent probe and a fluorescent quenching probe
A method for performing gene amplification and real-time monitoring of an amplification product on the A chip will be described with reference to specific examples. Further, from the dissociation curve of the amplified gene, the fluorescent probe and the fluorescent quenching probe, S
NPs were detected.

1) Experimental method (1) Fluorescent emission probe The fluorescent emission probe and the fluorescence quenching probe are shown in Table 13. These are the same base sequences as those used in Example 42. The fluorescent probe was used with its 3 ′ end phosphorylated. The method for synthesizing them is the same as in Example 42. The probe sequences and the modification positions in Texas red and Dabcyl probes are as shown in Table 10.

(2) Fluorescence Quenching Probe The base sequence of the fluorescence quenching probe is the same as the base sequence of the fluorescent probe. An MMT amino linker was introduced into the 5 'end of the fluorescence quenching probe using 5'-Amino-Modifier C12 (manufactured by Glen Research). The 3 'terminal base is Amino-Modifier C6 dC (Glen Research)
Was used to introduce a TFA amino linker. After deprotection of the protecting group TFA, the oligonucleotide was modified with BODIPY FL (Molecular probes) via an amino linker. The fluorescence quenching probe is in a state where the 3 'end is phosphorylated. The target nucleic acids shown in Table 12 were used. Other detailed purification methods and modification methods were the same as those in Example 8.

(3) Primer 5'CTTGGGGGGGCATATCTG as forward primer
Using a sequence that is 3 ', 5'A
CATCCGGCTTTGACTCTCTCT 3 'was used. This primer set can amplify a part (2509 bp) of the human CYP21 gene. Fluorescent and quenching probes are 100% of the corresponding amplification products without SNPs
It has a complementary sequence. Therefore, it was expected that the amount of change in the fluorescence intensity of the fluorescent emission probe and the fluorescence quenching probe shown in Table 13 would increase as the number of corresponding amplification products increased.

<Preparation of DNA Chip> One spot of each probe solution was spotted on a slide glass. Other methods for preparing the DNA chip are the same as those for preparing the DNA chip using the quenching probe.

When a fluorescent probe is immobilized on a slide glass, the Texas red fluorescence is quenched when the probe does not hybridize to the target nucleic acid, but when the probe hybridizes, the fluorescence emission is reduced. It is significantly higher than that without soybean. Conversely, when the fluorescence quenching probe is immobilized on the slide glass, BODIPY F
L emits fluorescence, but when it is hybridized, the fluorescence is more quenched than when it is not hybridized.

<Method of Real-Time Monitoring PCR> PCR was performed on a DNA chip using the above-mentioned primers with the human genome used in Example 29 as a template, and the PCR amplification product was immobilized on a fixed fluorescent probe or fluorescent quenching probe. Detected. The experiment was performed using the apparatus shown in FIG. On a DNA chip on which a fluorescent probe and a fluorescent quenching probe are immobilized,
Primer, template, Taq polymerase, dNTP, MgC
It was placed a solution, including l _2. The solution was covered with a cover glass so as not to leak, and the cover glass was sealed with nail polish. The chip was placed on a transparent heating plate incorporating a temperature control program, and a PCR reaction was performed on the chip. The amplified product was detected in real time by following the change in fluorescence between the immobilized fluorescent probe and the fluorescent quenching probe with the microscope shown in FIG.

The first Denature is performed at 95 ° C. for 120 seconds, and the PCR cycle is Denature = 95 ° C./60 seconds, anneal
The test was performed at aling = 60 ° C./60 seconds and extension = 72 ° C./120 seconds. The Primer concentration was 0.5 μM for both forward and reverse in the final concentration. The template is 1.5n
Added at a final concentration of g / μl. Gene as DNA polymerase
Taq ™ (Nippon Gene) was used at a concentration of 0.5 Units / 20 μl. The Mg ion concentration was 2 mM. dNTP was added to a final concentration of 2.5 mM each.

<Preparation of Dissociation Curve> A dissociation curve between the immobilized fluorescent probe and the fluorescence quenching probe and the PCR amplification product was prepared in the same manner as in Example 42, and SNPs were detected.

2) Results The experimental results are shown in FIG. From the figure, it can be seen that the amount of change in the fluorescence of all the probes increases as the number of cycles increases. Therefore, it was shown that the method of the present invention can simultaneously perform gene amplification and real-time detection of the amplified product. FIG. 39 shows the result of preparing a dissociation curve between the amplification product and each probe. As can be seen from the figure, as the temperature decreased, significant changes in fluorescence were observed for all the probes. This indicated that the fluorescence emitting probe and the fluorescence quenching probe hybridized with the target base sequence. Thus, it was found that the dissociation curve between the probe of the present invention and the target nucleic acid can be easily monitored. The dissociation curve between the amplification product and the fluorescence emission probe and fluorescence quenching probe of WIAF-10600 almost coincides with the dissociation curve between the artificially synthesized target containing no mismatch obtained in Example 42 and the WIAF-10600 probe. Thus, the human genome used as a template was shown to be 100% complementary to the probe sequence of WIAF-10600. The dissociation curve between the amplification product and the WIAF-10578 fluorescence emission probe and fluorescence quenching probe almost coincides with the dissociation curve between the artificially synthesized target containing the mismatch obtained in Example 42 and the WIAF-10578 probe. Therefore, it was shown that the human genome used this time contained a mismatch with the probe sequence of WIAF-10578. Thus, the DN of the present invention
It was found that the use of the A-chip enables simultaneous analysis of a plurality of types of SNPs in the amplified product after gene amplification.

[0338]

[0339]

The present invention has the following effects. 1) First invention (fluorescence emitting probe): The probe of the present invention is obtained by simply binding a fluorescent dye and a quencher substance to a single-stranded deoxyribooligonucleotide that does not form a stem loop. Designing the nucleotide sequence of a probe that hybridizes to a nucleic acid is not complicated and easy. In addition, before hybridization to the target nucleic acid, the background of the measurement is extremely low because the emission of the fluorescent dye is suppressed by the quencher substance. Thus, the measurement of the target nucleic acid is accurate. Moreover, it is simple,
Can be measured in a short time.

2) Second Invention (Fluorescence Quenching Probe): (1) The probe of the present invention is obtained by simply binding a specific fluorescent dye to a single-stranded deoxyribooligonucleotide. The fluorescence intensity is designed to decrease when the reaction system shifts from the hybridization system to the hybridization system. So the design of the probe is not complicated and easy. As a result, the measurement of the target nucleic acid is accurate and simple. (2) The fluorescent quenching probe of the present invention particularly composed of a chemically modified oligonucleotide or the like, or the fluorescent quenching probe of the present invention composed of a chimeric oligonucleotide or the like measures RNA having a complicated structure, particularly nucleic acid such as tRNA. It was developed for. According to the present invention, these nucleic acids can be easily, simply and accurately measured.

3) Third Invention (Invention Regarding Use of the Fluorescent Probe or Fluorescence Quenching Probe of the Present Invention) (1) When the fluorescent luminescent probe or fluorescent quenching probe of the present invention is used, it is contained or attached. In addition, a measurement kit for measuring the concentration of a target nucleic acid, and a nucleic acid chip or a nucleic acid device such as a DNA chip to which the probe is bound can be simply and easily manufactured. (2) When the probe, the measurement kit, the nucleic acid chip or the nucleic acid device of the present invention is used, the operation of removing the unreacted nucleic acid probe from the measurement system is not performed, so that the concentration of the target nucleic acid can be reduced in a short time. (3) When applied to a complex microbial system or a symbiotic microbial system, the amount of a specific strain in the system can be measured specifically and in a short time. (4) According to the present invention, analysis or measurement of polymorphism or mutation such as SNP of a target nucleic acid or gene becomes simple and accurate.

(5) The quantitative PCR method using the probe 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 conventionally known specific PCRs. b. In addition, since the specificity of PCR can be kept high, the amplification of the primer dimer is slowed, so that the quantification limit is reduced by about one order of magnitude as compared with 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.

(6) The present invention provides a real-time quantitative PC using the fluorescent probe and the quenching probe of the present invention.
The R method provides a data analysis method for analyzing the obtained data. (7) Then, when a calibration straight line for calculating the copy number of the nucleic acid is prepared for the nucleic acid sample of the unknown copy number using the data analysis method, the correlation coefficient of the calibration curve is smaller than that obtained by the conventional method. Very high. Thus, the use of the data analysis method of the present invention makes it possible to determine the exact copy number of a nucleic acid.

(8) 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 And real-time quantitative PC using it
The use of an R measuring or analyzing apparatus makes it possible to automatically create a calibration line having a high correlation coefficient.

(9) Further, 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, and a computer-readable recording medium recording the procedure of the analysis method as a program, and a real-time quantitative PCR measurement or analysis apparatus using the same, An accurate Tm value can be obtained.

(10) Quantitative polymorphism analysis method The amount of the target gene and the composition ratio of the polymorphism of the gene were determined by the following methods.
The target nucleic acid is amplified using the novel quantitative PCR method of the present invention, and the amplification is performed on the amplified nucleic acid. The amplified nucleic acid is labeled with a fluorescent dye. Therefore, since polymorphism analysis can be performed using a fluorescent dye as a marker, polymorphism analysis can be performed easily, quickly, and with good quantitativeness.

[Brief description of the drawings]

FIG. 1 shows changes in fluorescence intensity of a solution system when a target nucleic acid is added to a solution system containing a nucleic acid probe of the present invention. Horizontal axis: time (seconds), vertical axis: fluorescence intensity

FIG. 2 Calibration curve of target nucleic acid using nucleic acid probe of the present invention Horizontal axis: Target nucleic acid concentration, Vertical axis: Fluorescence intensity

Fig. 3 Fluorescent dye (Texas Red) and quencher substance (D
Probe design and target nucleic acid design to study the effect of the distance (base number) between a fluorescent dye and a quencher substance on the fluorescence emission rate of a fluorescent probe utilizing the interaction between abcyl).

Fig. 4 Fluorescent dye (Texas Red) and quencher substance (D
FIG. 7 is a diagram showing the effect of the distance (number of bases) between a fluorescent dye and a quencher substance on the fluorescence emission rate of a fluorescent probe utilizing the interaction between abcyl) and a fluorescent dye.

FIG. 5: Fluorescent dye (Texas Red) and quencher substance (D
abcyl) both probe design and target nucleic acid design in which the base in the strand of deoxyribooligonucleotide has been modified.

FIG. 6: Fluorescent dye (Texas Red) and quencher substance (D
abcyl), using a probe in which a base in the chain of a deoxyribooligonucleotide has been modified, to see the effect of the distance (base number) between a fluorescent dye and a quencher substance on fluorescence emission.

[FIG. 7] Using the nucleic acid probe obtained in Example 7, 335 to 358 counted 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. 8 shows the effect of heat treatment on the hybridization of a 35 base strand 2-O-Me probe to a target nucleic acid. Dotted line: Target nucleic acid was added after heat treatment of rRNA. Solid line: unheated rRNA

FIG. 9 shows the effect of the number of bases on the base chain of the probe, the helper probe and the modification of the methyl group at the 2′-carbon OH group of ribose at the 5 ′ end of the probe on the hybridization between the probe and 16S rRNA of the target nucleic acid.

FIG. 10 is a calibration curve for measuring rRNA according to the method of the present invention.

FIG. 11 shows the r of the combined culture system of the KYM7 strain and the KYM8 strain.
Analysis of the time course of RNA amount by the FISH method of the present invention.

FIG. 12 illustrates a DNA chip of the present invention.

FIG. 13 is a diagram illustrating devices for detecting and measuring SNPs using the DNA chip of the present invention.

FIG. 14 is a view showing experimental results of SNPs detection measurement using the DNA chip of the present invention.

FIG. 15: Primer 1 labeled with bodypy FL / C6
FIG. 6 is a diagram showing the relationship between the number of cycles and the amount of decrease in the emission of the fluorescent dye by the quantitative PCR method using Nos. And 2.

FIG. 16: Primer 1 labeled with bodypy FL / C6
FIG. 6 is a diagram showing the relationship between the number of cycles and the logarithmic value of the amount of decrease in the emission of the fluorescent dye, using the quantitative PCR method using Nos. And 2. ~ In the figure
The symbol “” has the same meaning as in FIG. 15.

FIG. 17 shows a calibration curve of Escherichia coli 16S rDNA prepared using the quantitative PCR method of the present invention. n: 10 ⁿ

FIG. 18 shows a case where real-time quantitative 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. It is a figure which shows the change of the reduction rate of fluorescence intensity. The figure below shows the number of cycles (thresholdnumbe
(r: Ct value) was calculated and a calibration curve was created.

FIG. 19 is a diagram showing a fluorescence reduction curve obtained by real-time quantitative PCR using a primer labeled with the bodypy FL / C6 of the present invention when the correction arithmetic processing of the present invention is not performed.

FIG. 20 is a diagram showing a fluorescence reduction curve obtained by real-time quantitative PCR in the same manner as in the case of the curve in FIG. 19, except that the value at the 10th cycle of each curve is corrected to 1.

FIG. 21 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.

FIG. 22 shows human genomic DNA obtained from the data shown in FIG.
FIG. y = human β-globin gene copy number x = cycle number (Ct) R ² = correlation coefficient

FIG. 23 is a view showing a curve obtained by plotting a value obtained by performing a correction operation on the measured value of each cycle in [Equation 1] in FIG. 19 with respect to each cycle number.

FIG. 24 is a view showing a curve obtained by plotting a value obtained by performing an arithmetic process on each cycle of FIG.

FIG. 25 is a diagram showing a curve obtained by plotting a value obtained by performing an arithmetic operation on the operation processing value of each cycle in FIG.

FIG. 26 is a graph showing Ct from each log (fluorescence change rate) value shown in FIG. 24;
0.1, 0.3, 0.5, 0.7,
FIG. 9 is a diagram in a case where 0.9 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.

FIG. 27. One and ten copies of human genomic DNA
FIG. 3 is a graph showing a fluorescence decrease curve when real-time quantitative PCR was performed using primers labeled with bodypi FL / C6 of the present invention. However, the correction calculation processing of [Equation 1] was performed.

FIG. 28 is a view showing a melting curve of a nucleic acid obtained by performing a melting curve analysis of the nucleic acid for the PCR amplification product shown in FIG. 27.

FIG. 29 is a view showing a curve indicating a Tm value obtained by differentiating the curve of FIG. 28 (the valley is the Tm value). 1: Control 2: Target nucleic acid = 1 copy 3: Target nucleic acid = 10 copies

FIG. 30 shows 16Sr using the quantitative PCR method of the present invention.
Amplification curve of RNA gene (cDNA).

FIG. 31 shows a calibration curve of cDNA prepared by the data analysis method of the present invention.

FIG. 32 shows the analysis pattern of T-RFLP of the polymorphism system of the present invention. bp: number of bases (base pair)

FIG. 33 shows the results of a quantitative PCR method using a fluorescent probe as a primer (fluorescent primer) (index graph).

FIG. 34 shows a calibration curve of 16S rRNA gene (fluorescent primer: 0 μM). A: Copy number of the artificial complex microbial system (about 296,000
copies)

FIG. 35 shows a result of real-time monitoring of a PCR amplification product in a real-time quantitative PCR method using a fluorescent primer and a calibration curve obtained. Each line in the figure corresponds to that in FIG.

FIG. 36 shows the results of SNPs detection using a fluorescent probe.

FIG. 37 shows the results of detecting SNPs using a DNA chip with a fluorescent probe immobilized thereon.

FIG. 38 shows the results of performing a real-time monitoring PCR method using a DNA chip to which a fluorescent probe and a fluorescent quenching probe were bound.

FIG. 39 shows P when PCR was performed using a DNA chip to which a fluorescent probe and a fluorescent quenching probe were bound.
Diagram of the melting curve of the CR product.

[Explanation of symbols]

1: Upright Olympus microscope (AX80 type) 2: Transparent heating plate for microscope (MP-10MH-PG, Kitasato Supply Co., Ltd.) 3: Temperature controller 4: Cooling CCD camera (C5810 type, Hamamatsu Photonics) 5 ： Image analysis device

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 C12N 15/00 ZNAA (72) Inventor Takahiro Kanakawa 1-1-1 Higashi, Tsukuba-shi, Ibaraki Independent Administration National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor Yoichi Kamagata 1-1-1 Higashi Tsukuba, Ibaraki Prefecture Independent Administrative Law National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor Masaki Torimura Higashi Tsukuba, Ibaraki 1-1-1 National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor Shinya Kurata 1-9-8 Higashikanda, Chiyoda-ku, Tokyo Environmental Engineering Co., Ltd. (72) Inventor Kazutaka Yamada Environmental Engineering Co., Ltd. 1-9-8 Higashi Kanda, Chiyoda-ku, Tokyo (72) Inventor Toyoichi Yokomaku F-term (reference) 1-9-8 Higashikanda, Chiyoda-ku, Kyoto 2G045 CB01 CB21 DA12 DA13 DA14 FA16 FA19 FB02 FB07 FB12 GC15 HA10 HA16 JA01 4B024 AA11 CA02 CA04 CA05 CA09 HA14 4B029 AA07 AA23 BB20 CC03 CC08 FA12 4B063 QA01 QA13 QQ42 QQ52 QR32 QR55 QR62 QS34 QX02

Claims (50)

[Claims] 1. A nucleic acid probe comprising a single-stranded oligonucleotide capable of hybridizing to a target nucleic acid and a fluorescent dye and a quencher substance being labeled, wherein the nucleic acid probe is hybridized to the target nucleic acid. As the fluorescence intensity of the hybridization reaction system increases,
A fluorescent dye and a quencher substance are labeled on the oligonucleotide, and the oligonucleotide does not form a stem-loop structure between the base where the fluorescent dye is labeled and the base where the quencher substance is labeled. A novel nucleic acid probe for nucleic acid measurement, characterized in that: 2. The novel nucleic acid probe for nucleic acid measurement according to claim 1, wherein the fluorescent dye and the quencher substance are labeled at the same nucleotide position of the single-stranded oligonucleotide. 3. The distance between the base where the fluorescent dye is labeled and the base where the quencher substance is labeled is 1 to 20, or Δ (any of 3 to 8) in terms of the number of bases. 2. The novel nucleic acid probe for nucleic acid measurement according to claim 1, wherein (integer) + 10n｝ (where n is an integer including 0). 4. The novel nucleic acid probe for nucleic acid measurement according to claim 1, wherein the single-stranded oligonucleotide has the same length as the target nucleic acid. 5. A nucleic acid probe labeled with at least one fluorescent dye, wherein, when hybridized to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces its luminescence, and the probe comprises: When the nucleic acid probe is labeled with a fluorescent dye at a portion other than the 5′-terminal phosphate group or the 3′-terminal OH group of ribose or deoxyribose at the 3′-position, and the nucleic acid probe hybridizes to the target nucleic acid, The base sequence of the probe is designed so that one to three bases are separated from the base of the modified portion (the base of the modified portion is counted as 1) and at least one base of G (guanine) is present in the base sequence of the target nucleic acid. When a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid, the fluorescent dye reduces its emission. A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid. 6. A nucleic acid probe labeled with at least one fluorescent dye, wherein, when hybridized to a target nucleic acid, the fluorescent dye is a nucleic acid probe that reduces its luminescence, and the probe comprises: The 5′-terminal phosphate group or the 3′-terminal ribose or deoxyribose is labeled with a moiety other than the OH group at the 3′-position of the fluorescent dye, and when the nucleic acid probe is hybridized to the target nucleic acid, A plurality of base pairs of the probe-nucleic acid hybrid form at least one pair of G (guanine) and C (cytosine) at a distance of 1 to 3 bases from the base of the modified portion (the base of the modified portion is counted as 1). The nucleotide sequence of the probe is designed so that the nucleic acid probe labeled with a fluorescent dye hybridizes to the target nucleic acid. A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid, wherein the fluorescent dye reduces the emission of the fluorescent dye when activated. 7. When 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 the emission thereof, and the probe has a 5′-terminal. 3 'of phosphate group and 3' terminal ribose or deoxyribose
When the nucleic acid probe is hybridized to the target nucleic acid at both ends thereof, the nucleic acid probe is labeled 1 to 3 from the terminal base of the target nucleic acid hybridized to the probe.
The base sequence of the probe is designed such that the base pairs of the probe-nucleic acid hybrid form at least one pair of G (guanine) and C (cytosine) apart from each other (counting the terminal base as 1). When the nucleic acid probe labeled with a fluorescent dye is hybridized to the target nucleic acid, the fluorescent dye reduces the emission thereof, and the nucleic acid labeled with a fluorescent dye for measuring the concentration of the target nucleic acid. probe. 8. A nucleic acid probe comprising a 3′-carbon hydroxyl group of 3′-terminal ribose or deoxyribose, or 3 ′
The nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to any one of claims 1 to 7, wherein a hydroxyl group at the 3 'or 2' carbon of the terminal ribose is phosphorylated. 9. The 5 ′ end and / or 3 ′ of a nucleic acid probe
The terminal phosphate group is labeled with a fluorescent dye.
8. A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to any one of 7. 10. The oligonucleotide of a nucleic acid probe for measuring a nucleic acid is a chemically modified nucleic acid.
A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to any one of the above. 11. The nucleic acid which has been chemically modified is 2′- O -methyl oligonucleotide, 2′- O -ethyl oligonucleotide, 2′- O -butyl oligonucleotide, 2 ′
- O - ethylene oligonucleotide, or 2'O - benzyl oligonucleotide nucleic acid probe labeled with a fluorescent dye for concentration measurement of the target nucleic acid of claim 10. 12. The oligonucleotide of a nucleic acid probe for nucleic acid measurement, wherein the oligonucleotide comprises a chimeric oligonucleotide containing ribonucleotides and deoxyribonucleotides.
A nucleic acid probe labeled with a fluorescent dye for measuring the concentration of a target nucleic acid according to any one of claims 1 to 11, which is a ligonucleotide). 13. The chimeric oligonucleotide,
The target nucleic acid according to claim 12, wherein the target nucleic acid is a 2'- O -methyl oligonucleotide, a 2'- O -ethyl oligonucleotide, a 2'- O -butyl oligonucleotide or a 2'- O -benzyl oligonucleotide. Nucleic acid probe labeled with a fluorescent dye for concentration measurement. 14. A method for measuring the concentration of a target nucleic acid, comprising hybridizing the nucleic acid probe according to claim 1 to a target nucleic acid and measuring the fluorescence intensity of a measurement system. 15. A nucleic acid probe for nucleic acid measurement according to any one of claims 1 to 13, which is hybridized to a target nucleic acid, and a change in luminescence of a fluorescent dye before and after hybridization is measured. The method for measuring the concentration of the target nucleic acid to be performed. 16. A method for measuring the concentration of a target nucleic acid using the nucleic acid probe according to any one of claims 10 to 13 under conditions suitable for sufficiently destroying the higher-order structure of the target nucleic acid. A method for measuring the concentration of a target nucleic acid, comprising subjecting the target nucleic acid to a heat treatment and then hybridizing the nucleic acid probe and the target nucleic acid. 17. A helper probe for performing a hybridization reaction is added to a hybridization reaction system before the hybridization reaction.
2. The method for measuring the concentration of a target nucleic acid according to item 1. 18. A polymorphism or / and / or a target nucleic acid.
And a method for analyzing or measuring mutation, wherein the nucleic acid probe according to any one of claims 1 to 13 is hybridized to a target nucleic acid, and the amount of change in fluorescence intensity is measured. A method for analyzing or measuring a polymorphism and / or mutation of a nucleic acid. 19. Amplifying a target gene by a quantitative gene amplification method and performing polymorphism analysis on the target gene to measure the amount of the target gene, the composition ratio of the polymorphism of the gene, or the amount of each component. A novel quantitative polymorphism analysis method characterized by the following. 20. Polymorphism analysis is performed by T-RFLP (terminal restrict).
ion fragment length polymorphism), RFLP (restrictio
n fragment length polymorphism) method, SSCP (single s
trand conformation) method or CFLP (cleavage fragm
The quantitative polymorphism analysis method according to claim 19, which is an ent length polymorphism) method. 21. A method for quantitative gene amplification, comprising:
The quantitative polymorphism analysis method according to claim 19 or 20, which is an R method. 22. The method for quantitative polymorphism analysis according to claim 21, wherein the quantitative PCR method uses the nucleic acid probe according to any one of claims 1 to 13. 23. The method for quantitative PCR according to claim 1, wherein
The quantitative polymorphism analysis method according to claim 21, wherein the amount of change in luminescence of the fluorescent dye is measured using the nucleic acid probe according to any one of (1) to (13) as a primer. 24. The quantitative polymorphism analysis method according to claim 21, wherein the quantitative PCR method is a real-time monitoring quantitative PCR method. 25. A kit for measuring the concentration of a target nucleic acid, the kit for measuring the concentration of a target nucleic acid, comprising or accompanying the nucleic acid probe according to any one of claims 1 to 13. 26. The kit for measuring the concentration of a target nucleic acid according to claim 25, wherein the kit contains or is accompanied by a helper probe. 27. A measurement kit for analyzing or measuring a polymorphism and / or a mutation of a target nucleic acid, wherein the kit is for analysis or measurement.
A kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, comprising the nucleic acid probe according to any one of the above. 28. The measurement kit for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid according to claim 27, which contains or is associated with a helper probe. 29. A reagent kit used for a quantitative PCR method in the quantitative polymorphism analysis method according to any one of claims 19 to 24, wherein the reagent kit according to any one of claims 1 to 13 is used. A reagent kit for quantitative PCR, comprising or attached to a nucleic acid probe. 30. A plurality of nucleic acid probes according to any one of claims 1 to 13, which are bound to the surface of a solid support, and a target nucleic acid is hybridized to a corresponding nucleic acid probe to reduce the amount of change in fluorescence intensity. A device for measuring the concentration of at least one target nucleic acid among a plurality of types of nucleic acids, wherein the concentration of the target nucleic acid can be measured by measuring. 31. The device for measuring nucleic acids according to claim 30, wherein the nucleic acid probes are arranged in an array on the surface of a solid support, and each of them measures the concentration of one or more nucleic acids. 32. For each nucleic acid probe bound to the surface of the solid support, at least one temperature sensor and a heater are installed on the opposite surface so that the corresponding nucleic acid probe binding region has an optimal temperature condition. 32. A device for measuring the concentration of at least one target nucleic acid of the plurality of nucleic acids according to claim 30 or 31, which can be temperature-controlled. 33. A concentration measurement of at least one target nucleic acid among a plurality of types of nucleic acids, wherein the target nucleic acid is measured using the nucleic acid measurement device according to claim 30. Method. 34. A method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid, comprising using the nucleic acid measuring device according to claim 30. 35. A method for quantitative polymorphism analysis, comprising using the device for nucleic acid measurement according to claim 30. 36. The target nucleic acid according to any one of claims 14 to 17, wherein the target nucleic acid is a nucleic acid contained in cells derived from microorganisms or animals obtained by pure isolation or contained in a homogenate of those cells. 3. The method for measuring the concentration of a target nucleic acid according to item 1. 37. The method for analyzing or measuring a polymorphism and / or mutation of a target nucleic acid according to claim 18, wherein the target nucleic acid is a nucleic acid of a complex microbial system or a symbiotic microbial system in a cell or a cell homogenate. 38. The quantitative polymorphism analysis method according to claim 19, wherein the target nucleic acid is a nucleic acid of intracellular or cell homogenate of a complex microorganism system or a symbiotic microorganism system. 39. The PCR method according to claim 1, wherein
Performing a reaction using the nucleic acid probe according to any one of the above, and measuring the initial concentration of the amplified target nucleic acid from the change rate of the fluorescence intensity value generated by hybridization between the probe and the amplified target nucleic acid. Featured PC
A method for measuring the concentration of a target nucleic acid used in the R method. 40. The PCR method according to claim 1, wherein
A reaction is performed using the nucleic acid probe according to any one of the above as a primer, and the target amplified from the change rate of the fluorescence intensity value generated by hybridization of the primer or the amplified nucleic acid amplified from the primer with the amplified target nucleic acid A method for measuring a target nucleic acid using a PCR method, comprising measuring an initial concentration of a nucleic acid. 41. The PCR method according to claim 1, wherein
The reaction is carried out using the nucleic acid probe according to any one of the above, a reaction system in which the probe is decomposed and removed by a polymerase during a nucleic acid extension reaction or a reaction system in which a nucleic acid denaturation reaction is completed or a nucleic acid denaturation reaction is completed. PCR, comprising measuring a fluorescence intensity value and a fluorescence intensity value of the reaction system when the target nucleic acid or the amplified target nucleic acid is hybridized with the nucleic acid probe, and calculating a change rate of the fluorescence intensity value from the former. Measuring the initial concentration of the target nucleic acid amplified by the method. 42. The PCR method according to claim 1, wherein
The reaction is performed using the nucleic acid probe according to any one of the above as a primer, the fluorescence intensity value of the reaction system in which the probe and the target nucleic acid or the amplified target nucleic acid are not hybridized and the nucleic acid probe is the target nucleic acid or the amplified target nucleic acid. A method for measuring an initial concentration of a target nucleic acid amplified by PCR, wherein a fluorescence intensity value of a reaction system during hybridization is measured, and a decrease rate of the fluorescence intensity value of the former is calculated. 43. The PCR method is a real-time quantitative PC
43. The method for measuring an initial concentration of a target nucleic acid amplified by PCR according to claim 41 or 42, which is an R method. 44. The method for analyzing data obtained by the nucleic acid measurement method according to any one of claims 14 to 24 or 33 to 43, wherein the target nucleic acid is a fluorescent dye. The fluorescence intensity value of the reaction system after hybridization with the labeled nucleic acid probe is corrected by the fluorescence intensity value of the reaction system obtained after the probe-nucleic acid hybrid complex thus formed is dissociated. A data analysis method for measuring the concentration of a target nucleic acid. 45. The method for analyzing data obtained by the real-time quantitative PCR method according to claim 43, wherein the amplified nucleic acid in each cycle is combined with a fluorescent dye or the amplified nucleic acid is labeled with a fluorescent dye. The fluorescence intensity value of the reaction system after hybridization with the nucleic acid probe thus obtained is determined by the fluorescent dye-nucleic acid complex formed in each cycle or the probe thus formed.
Data analysis for a real-time quantitative PCR method, comprising an arithmetic processing step for correcting with a fluorescence intensity value of a reaction system obtained after dissociation of a nucleic acid hybrid complex (hereinafter referred to as a correction arithmetic processing step). Method. 46. The data analysis method for a real-time quantitative PCR method according to claim 45, wherein the correction operation processing step according to claim 45 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} : after the amplified nucleic acid binds to the fluorescent dye or after the amplified nucleic acid hybridizes with the nucleic acid probe labeled with the fluorescent dye in the nth cycle Fluorescence intensity value of the reaction system, f _{den, n} : fluorescence intensity value of the reaction system after dissociation of the formed fluorescent dye-nucleic acid complex or formed probe-nucleic acid hybrid complex in the nth cycle] 47. The method according to claim 46, wherein the data obtained by the real-time quantitative PCR method according to claim 43 is analyzed, wherein the correction calculated by [Equation 1] or [Equation 2] in each cycle. Substituting the calculated value into the following [Equation 3] or [Equation 4], calculating a fluorescence change ratio or a fluorescence change ratio between samples in each cycle, and comparing the calculated values. Data analysis method for the method. F _n = f _n / f _a [Equation 3] F _n = f _a / f _n [Equation 4] wherein, F _n: the n th cycle, calculated by [Equation 3] or [Equation 4] Fluorescence 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 observed Any number of cycles before 48. The method according to claim 47, wherein the data obtained by the real-time quantitative PCR method according to claim 43 is analyzed. 1) The change in fluorescence calculated by [Equation 3] or [Equation 4]. ratio or by using the data of the fluorescence change rate, [equation 5], the process of arithmetic processing by the [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｝ [Formula 6] log _b ｛(F _n -1) × A｝, ln ｛(F _n -1) × A｝ [ [Formula 7] [where, A and b: arbitrary numerical values, _Fn : the ratio of change in fluorescence or the ratio of change in fluorescence in the nth cycle calculated by [Formula 3] or [Formula 4]], 2) the above 1) An arithmetic processing step for calculating the number of cycles in which the arithmetic processing value has reached a certain value; Calculation process for calculating the relational expression between the cycle number in the nucleic acid sample and the copy number of the target nucleic acid at the start of the reaction, and 4) the calculation process for calculating the copy number of the target nucleic acid at the start of the PCR in the unknown sample. A data analysis method for a real-time quantitative PCR method characterized. 49. Performing PCR on the target nucleic acid using the nucleic acid probe according to any one of claims 1 to 13,
A method for analyzing a melting curve of a target nucleic acid, wherein the melting curve of a target nucleic acid is analyzed to determine a Tm value of each amplified nucleic acid. 50. The data analysis method for a real-time quantitative PCR method according to any one of claims 45 to 48, wherein the nucleic acid amplified by the PCR method is cooled from a low temperature until the nucleic acid is completely denatured. A process of gradually increasing the temperature, a process of measuring the fluorescence intensity at predetermined time intervals in this process, a process of displaying the measurement result on the display as a function of time and drawing a melting curve of the nucleic acid on the display, The value obtained by differentiating the curve (−dF /
dT, F: fluorescence intensity, T: time), displaying the differentiated value as a differential value on a display, and obtaining an inflection point from the differential value. Analysis method for dynamic PCR method.