JP2000166598A - Method and apparatus for quantitatively analyzing target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a constitutionally simple method and apparatus for quantitatively analyzing a target nucleic acid. SOLUTION: The purpose of this invention is achieved by the following steps: (1) mixing a biological sample including a target nucleic acid with primers, a fluorescently labeled substrate and a DNA polymerase to form a test liquid; (2) performing a preferred reaction for amplifying the nucleic acid and then making the test liquid adhere to the surface of a sliding glass 13 in a point-like state, followed by putting the glass 13 along with the test liquid on a specimen support 12 of a fluorescence inverted microscope; (3) calculating data measured by a photomultiplier 2 with a data-processing unit 3 using an auto-correlation function and displaying on a display device 4 quantitative information digitized or graphed with respect to the target nucleic acid based on the calculated result.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for analyzing a nucleic acid molecule present in a sample, and more particularly to an analysis method and apparatus for detecting a target nucleic acid molecule based on a result of a nucleic acid amplification reaction. INDUSTRIAL APPLICABILITY The present invention is applied to a detection method that obtains the presence / absence and / or reaction amount of an amplification reaction by effectively using tracking data on a single nucleic acid molecule. Therefore, the present invention can be effectively applied to any purpose of obtaining information related to the number of target nucleic acid molecules.

[0002]

2. Description of the Related Art Detection of nucleic acids such as DNA and RNA and measurement of molecular weight are the most important analytical tools in biochemical and molecular biology research. Recently, it has become an important analytical tool in gene diagnosis and gene therapy. For example, in the PCR (polymerase chain reaction) method, which is very important as a gene amplification method, the final detection of the amplified gene is performed by gel electrophoresis and fractionation by chain length (molecular weight). After separation, the presence or absence of fractionation at a specific position is measured.

[0003] A DNA polymerase as a nucleic acid synthase, a primer for initiating replication, and four types of bases (dATP, dGTP, dTTP,
When the target nucleic acid molecule is treated in the presence of dCTP) under conditions that allow the polymerization reaction of the substrate molecule, a single-stranded DNA derived from the target nucleic acid is used as a template (template DNA).
It is known that nucleic acid amplification occurs such that a complementary strand is synthesized to form a double strand (dsDNA).

In the PCR method, amplified dsDNA
Is measured by a fluorimeter as the total amount of fluorescence emitted by the entire test solution in an appropriate container or on a plate. The total amount of fluorescence thus measured corresponds to the target DNA after being sufficiently amplified. However, the amplification reaction in PCR is a reaction process under opposite temperature conditions, that is, dsDN.
Heating the sample at elevated temperature to denature A to separate it into two separate ssDNAs;
And a step of lowering the temperature in order to hybridize with the above-mentioned pair of primers, it is difficult to quantitatively track the state of amplification. Therefore, in the conventional PCR method, it was impossible to quantitatively analyze the total amount of the target nucleic acid during or before amplification.

In recent years, the fluctuation motion of a labeled molecule in a liquid has been measured, and an autocorrelation function (Autocorrelation function) has been measured.
A technique has emerged that uses a function to accurately measure the micromotion of each target molecule. In this type of method, in particular, a fluorescent molecule as a labeling molecule is measured by an optical instrument, so that correlated fluorescence spectroscopy (Fluoresen) is used.
ce Correlation Spectrosco
py (FCS for short). A method of calculating measurement data on a biological material using FCS is reported by using FCS in a hybridization reaction between a labeled nucleic acid probe and a target nucleic acid molecule (Kinj
o, M. Rigler, R .; , Nucleic Ac
ids Research, 23, 1795-179
9, 1995).

[0006] There have recently been several reports of the detection of target genes using FCS. NASBA (Nucleic
AcidSequenceBasedAmplific
) is bound to FCS, and HIV (H
humanImmunodeficiencyViru
s) (Oe)
hlenschlager, F .; Proc. Nat
l. Acad. Sci. USA, 93, 12811-1
2816, 1996). This NASBA method is a modification of PCR, but it includes T7 RNA polymerase, reverse transcriptase,
And RNaseH, which require two primers and another fluorescently labeled primer as a probe.

A method simpler than the NASBA method (amplification probe extension detection method by FCS, APEX (Amplif
iedProbeExtension) has been reported from the same group (Walter, NG et al., Pr.
oc. Natl. Acad. Sci. USA, 93, 1
2805-12810, 1996). Nevertheless, the forward primer and the reverse primer
e) In addition to the primer set, a separate fluorescently labeled primer is required as a probe. In this APEX experiment, a temporal shift of the autocorrelation function is observed after 26 cycles.

[0008]

However, the above N
Since both the ASBA method and the APEX method require a labeled probe different from the usual nucleic acid amplification material, the reaction conditions are complicated, and it is difficult to stably control the analysis accuracy. In addition, since the labeling molecule is labeled with one fluorescent label, of the probes labeled with the labeling molecule, the signal derived from the label emitted from the non-hybridizing probe significantly lowers the S / N ratio. The lower the initial concentration of the target nucleic acid molecule, the lower the detection sensitivity tends to be. In addition, many PCR
You have to turn the cycle.

An object of the present invention is to provide an analysis method and apparatus capable of accurately and simply quantifying the abundance of a target nucleic acid molecule in view of the actual situation of such a nucleic acid detection method. Another object of the present invention is to enable quantification with high sensitivity even with a small copy number. Another object of the present invention is to enable detection of the presence or absence of a target nucleic acid molecule even in a small amount of sample. Another object of the present invention is to make it possible to know the amplification process in PCR at a molecular level.

[0010]

SUMMARY OF THE INVENTION The present invention provides a fluorescent molecule-labeled nucleotide, fluor.
Escein-11-dUTP was prepared using a nucleic acid-containing sample and DNA.
It is based on the discovery that, when mixed with a polymerase and nucleic acid amplification was performed, it was possible to prove by image analysis that the movement state of the fluorescent dye molecules incorporated during the amplification process was changed. Further, the present invention is based on the finding that when the motion state of a fluorescent dye molecule is evaluated using an autocorrelation function, a result having a quantitative property can be obtained. Furthermore, the present invention is that by separating a labeled substrate molecule that has not been incorporated into the target nucleic acid molecule in the nucleic acid amplification process by a gel filtration column or the like, there is no problem in the measurement, and a further increase in sensitivity can be achieved. It is based on discovery.

That is, the method for analyzing a target nucleic acid according to the present invention is a method for analyzing a target nucleic acid utilizing a nucleic acid amplification reaction using a test solution containing a primer, a substrate molecule, a nucleic acid synthetase and a target nucleic acid molecule. Performing a nucleic acid amplification reaction with a test solution containing a substrate molecule labeled with a label molecule capable of generating a signal, and measuring a signal from the labeled molecule with respect to the nucleic acid-amplified test solution. Evaluating the ease of movement of the labeled molecule in the liquid based on the signal,
Quantifying the target nucleic acid molecule based on the evaluation result.

Here, the measuring step preferably includes a step of measuring the abundance of the labeled molecule in a predetermined measuring region.
Further, in this measurement step, it is preferable to measure the movement amount within a predetermined time a plurality of times. Further, between the step of performing the amplification reaction and the measurement step, the method further includes a step of removing the labeled substrate molecule that has not been involved in the nucleic acid amplification reaction, and the measurement step is performed without contacting the test solution. In this case, the sensitivity can be easily increased, and the measurement is non-contact. Therefore, the measurement accuracy is not reduced and the result with good reproducibility can be maintained.

Preferably, the evaluation step includes a step of converting the measured data into statistical data expressing a change in the movement amount. Further, the step of converting preferably includes a step of performing an operation by an autocorrelation function. In addition, the quantification step preferably includes a step of determining the presence or absence of a labeled molecule incorporated during nucleic acid amplification based on the evaluation result. Preferably, the quantification step includes a step of determining the number of target nucleic acid molecules incorporated during the nucleic acid amplification based on the evaluation result.

The apparatus for analyzing a target nucleic acid according to the present invention is characterized in that a target nucleic acid is amplified by a nucleic acid amplification reaction using a test solution containing a primer, a substrate molecule, a nucleic acid synthase (= polymerase or reverse transcriptase) and a target nucleic acid molecule. An analysis device, a holding unit for holding a test solution containing a substrate molecule labeled with a label molecule capable of generating a measurable signal, and for the held test solution, a signal from the label molecule after a nucleic acid amplification reaction. Measuring means for measuring, evaluation means for evaluating the ease of movement of the labeled molecule in a liquid based on the measured signal, and data output means for outputting data quantifying the target nucleic acid molecule based on the evaluation result. It is characterized by having.

Here, it is preferable that the measuring means has an optical system for performing a measurement within a minute visual field brought to a diffraction limited area near the focal point. Alternatively, it is preferable that the measuring means include a microscope that performs measurement in a microscopic field formed by the confocal optical system. Further, the diffraction limited area is preferably formed by an aperture having an average diameter of 30 ± 20 μm. Further, the diffraction limited region is preferably formed by an aperture having an average diameter of 20 ± 10 μm.

The minute visual field has an average radius of 200 ± 50.
It is preferably a substantially columnar region having a mean length of 2000 ± 500 nm on the optical axis.

It is preferable that the evaluation means include means for storing a plurality of measurement data obtained within a predetermined time, and calculation means for calculating the stored plurality of measurement data by an autocorrelation function. Further, the evaluation means has means for storing measurement data on a plurality of label molecules obtained in a predetermined measurement region, and calculation means for calculating the stored measurement data for each label molecule by an autocorrelation function. Is preferred.

Preferably, the data output means includes conversion means for converting, based on the result calculated by the autocorrelation function, to statistical data expressing a temporal change in a plurality of tracking data.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, at least one of substrate molecules incorporated in a nucleic acid amplification process in a PCR method.
The type is labeled with a label molecule that can generate a measurable signal. The substrate molecule having such a labeled molecule is in a free state without binding during the nucleic acid hybridization reaction step between the target nucleic acid and the primer for starting synthesis. In a nucleic acid synthesis reaction following a hybridization reaction, when a nucleic acid synthetase acts on a primer bound to a target nucleic acid molecule, the primer attempts to extend and successively takes in substrate molecules necessary for synthesis. Labeled molecules thus taken up with substrate molecules during nucleic acid amplification are less mobile in liquids than free labeled molecules.

Therefore, at least a part of the sample-containing test solution which has undergone an effective number of PCR cycles is subjected to the measurement of the labeled molecule while being quantitatively collected or held in a container or the like. Here, the test solution holding means is a test tube, a well,
It may be a concave container such as a petri dish or a flat plate-like support such as a slide glass. In some cases, it may be held by being spotted directly on the surface of the measuring means.

The measurement technique may be appropriately selected as long as it measures the change in the size of the labeled molecule accompanying the amplification. Gel electrophoresis, capillary electrophoresis, a flow cytometer for fluorescence measurement, an image cytometer, A fluorescence microscope or the like can be used effectively. By performing the measurement step in a three-dimensional microscopic visual field, free micromotion of the target molecule in the sample-containing solution can be measured with high accuracy in a natural state according to the result of the PCR reaction. On the other hand, when the measurement is performed in a two-dimensional field of view, unlike the Brownian motion, the three-dimensional free movement of the labeled molecule cannot be completely captured, so that the measurement accuracy is low. In forming a microscopic field of view, it can also be obtained by optically designing the light from the halogen lamp to converge or to be emitted from an aperture (pinhole, optical fiber end face, etc.) having an extremely small average radius. It is preferable to use convergent light by a laser beam.

Since the microscopic field in the measuring step is formed by the confocal optical system, measurement data with a large depth of field can be obtained. Position and output data can be supplied to the measuring means.

Since the microscopic field is a diffraction limited area near the focal point, individual labeled molecules can be measured with a high S / N ratio.

Since the diffraction limitation is formed by a pinhole having an average diameter of 15 ± 5 μm, measurement data from a small number of selected labeled molecules can be efficiently obtained.

Since the micro-area is a substantially cylindrical area having an average radius of 200 ± 50 nm and an average length of 2000 ± 500 nm on the optical axis, free micro-motion of the labeled molecule aimed in the measurement visual field can be efficiently obtained. can do.

In the present invention, the velocity of movement of a labeled molecule entering and exiting a minute measurement field of view is measured by using an index of increase or decrease or disappearance of an output intensity measured from one or more individual labeled molecules in a predetermined space. I have. Therefore, it is preferable that the type of the labeling molecule for labeling the substrate molecule is a labeling material that maintains a substantially constant output at a plurality of measurement points. As a material of such a labeling molecule, a luminescent substance,
Fluorescent substances, magnetic substances, radioactive substances and the like can be mentioned. In particular, as the luminescent substance and the fluorescent substance, it is preferable to select a dye that emits light or emits light for a long time. If a molecule having a luminescent dye or a fluorescent dye is used as the labeling molecule, measurement at the molecular level can be performed with an optically easy configuration. Examples of the fluorescent dye include DAPI, FITC, rhodamine, Cy3, CY3.5, Cy5, Cy5.5, Cy7.
And various known ones.

When the measurement step measures the ease of movement of the fluorescent molecules in the liquid, the fluorescent data can be received by a fluorescent measuring means such as a photomultiplier or a photodiode. It is more advantageous for the fluorescence measurement means to have a measurement mode capable of measuring a single photon, in order to perform individual measurements on fluorescent molecules.

In the case where the measuring step is to measure the fluctuation motion of the labeled molecule in the liquid, the present invention can be a more effective embodiment, and the micro motion of each target molecule can be accurately measured. can do. In measuring the fluctuation motion, an autocorrelation function (Autocorrela) was used.
It is preferable to perform the calculation by using a function. In particular, when fluorescence is used as a labeling molecule, autocorrelation fluorescence spectroscopy (Fluorescence C)
It is preferable to employ orlation spectroscopy (FCS for short). A method of calculating measurement data on a biological material using FCS is reported by using FCS in a hybridization reaction between a labeled nucleic acid probe and a target nucleic acid molecule (Kinj
o, M. Rigler, R .; , Nucleic Ac
ids Research, 23, 1795-179
9, 1995).

FIG. 1 schematically shows an apparatus for performing the FCS. The FCS apparatus receives an inverted fluorescence microscope 1 using a confocal optical system, a photomultiplier 2 for measuring fluorescence from a sample, and measurement data, and performs calculation by an autocorrelation function to perform numerical calculation or A data processing device 3 for performing graphing and a display device 4 for displaying calculation results on a screen
And

As shown in FIG. 1, the sample-containing liquid 11 can be easily set by being spotted on a slide glass 13 placed on a sample table 12. In this apparatus, in particular, since a small amount of the sample-containing liquid 11 is used, a lid 14 for preventing evaporation of water is put on the slide glass 13. Preferably, the lid 14 has a light transmittance as low as possible, so that the airtightness and the light shielding property can be obtained at the same time. However, it is preferable to use an inner surface of the lid having as low a light reflectivity as possible so as to prevent reflection of the excitation light.
An objective lens 15 set so as to be focused in the sample-containing liquid 11 is disposed immediately below the slide glass 13 where the sample-containing liquid 11 is located.

The fluorescence microscope 1 may be of an epi-illumination type.
In the epi-illumination type, the sample-containing liquid 11 may be directly spotted on the lower surface of the objective lens 15. The laser generator 16 as a light source of the fluorescence microscope 1 uses an argon (Ar) ion laser in FIG. 1, but a krypton argon (Kr-Ar) ion laser, a helium neon (He-Ne) laser,
A helium cadmium (He-Cd) laser or the like may be variously changed. Various operations such as loading and unloading of the slide glass 13, spotting of the sample-containing liquid onto the slide glass 13, and opening and closing of the lid 14 in the fluorescence microscope 1 may be appropriately automated as necessary.

FIG. 2 is an enlarged view showing a measurement portion of the fluorescence microscope 1 of FIG. In FIG. 2A, a small visual field region 20 is formed by the positional relationship between the slide glass 13 and the objective lens 15 having a predetermined numerical aperture (FA = 1.2 in the figure). As shown in FIG. 2B, the microscopic field region 20 actually has a substantially cylindrical visual field extending vertically from the focal point of the laser beam having a volume (in the figure, a narrow part in the middle). are doing. The visual field region 20 is defined by a length Z on the optical axis and an average radius W with the focus as a reference position. In such a fluorescence measurement in the minute region 20, the noise derived from the fluorescent molecules other than near the focal point in the sample-containing liquid 11 is effectively reduced by reducing the fluorescence to a minimum region where the minute movement of the fluorescent molecules can be traced. To contribute to accurate measurement of each fluorescent molecule.

The present invention is based on the various embodiments described above, and is based on the diagnosis of genetic diseases, paternity testing, criminal investigation, gene therapy,
It can be applied to molecular biological research, drug development, etc. The embodiments described above also include modifiable inventions without departing from the scope of the present invention. For example,
In the above-described embodiment, a measurement device that can be measured on a sample table of a fluorescence microscope under the same environment as that at the time of microscope observation has been described. However, the device disclosed in European Patent Publication No. 640828A1 is improved. Then, if the laser light emitted from the objective lens is optically designed so that the laser light emitted from the objective lens can be focused on the sample-containing liquid inside the thermal cycler, without transferring the sample-containing liquid onto the slide glass,
The amplification reaction can be measured continuously. In addition, the method of taking a plurality of data within the elapsed time in the PCR cycle is either a case where a plurality of data is obtained continuously or intermittently within a certain time after a predetermined cycle or a case where an appropriate number of measurement data is obtained for each different cycle. possible.

Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to these embodiments, and based on the gist of the present invention, an obvious range according to the technical level at the time of filing the present invention. Various changes in are possible.

[0035]

EXAMPLES (1) PCR in the presence of a fluorescently labeled nucleoside 0.1 Uμl ^-1 Pol type I DNA synthase (Ampl
iTaq Gold, Perkin-Elmer), 1
X Buffer I (Perkin-Elmer), 200
μM dATP, dGTP and dCTP, 120 μM
M dTTP, 80 μM fluorescein 11-dU
TP (FluoroGreen, Amersham F
lu-dUTP), and 0.8 μM forward (fo
(reverse) and a reverse solution (reverse) were used as a test solution.
In μl, standard nucleic acid amplification was performed.

As a template, 200 pg / 5 μl of full-length lambda phage DNA (48.5 kb) was used. Target DN
The length of A was 4000 bp. The nucleotide sequence of the primer used for nucleic acid amplification was GATGAG
TTCGGTGCCGTACAACT (base number = 71
31-7153), the reverse primer is CTTAACCA
GTGCGCTGAGTGTACT (base number = 1109
8-11120).

A PCR device for performing a nucleic acid amplification reaction is as follows:
Programmable thermal control system (PC700,
Astec), and an oil-free reaction tube (Takara Shuzo) was used as a PCR container for accommodating each of the above test solutions. That is, first, a metamorphic treatment at 96 ° C. for 15 minutes (initial metamorphic treatment)
, An annealing reaction at 55 ° C. for 15 minutes, an elongation reaction at 65 ° C. for 4 minutes, and a denaturation treatment at 96 ° C. for 1.5 minutes.
A CR cycle was performed. The PCR container was taken out in a predetermined PCR cycle and stored at -20 ° C until the next processing.

To remove unincorporated nucleosides, distilled water was added so that the mixture of each test solution subjected to PCR was 50 μl, and the mixture was added to a MicroSpin column (S-400HR, Pharmacia Biote).
c company). When the volume Vc of each solution collected by this column was measured by weight, the volume Vc was 55 μl to 60 μl.
l. The solution was used without further purification
The following FCS measurement was performed. The molecular weight of one base pair is 66
It was assumed to be 0 Da. Therefore, the molecular weight of lambda phage DNA was calculated to be 32 × 10 ⁶ Da.

(2) FCS Measurement FCS measurement was performed using an FCS device as shown in FIG. That is, a part of the test solution subjected to the nucleic acid amplification reaction is used as a sample, and the sample droplet is spotted on a sample slide, and a commercially available FCS device (ConfoCor, CarlZ) is used.
issJeNaGmbH) on a sample stage, magnification 40
Times objective lens (C-Apochromat, NA =
1.2), the light is excited by the CW Ar ⁺ laser beam that has passed through the lens, and the emitted light is SPCM-200-PQ (EG &), which is an avalanche diode (APD).
(Company G) to measure the fluorescence signal in single photon counting mode. The measured fluorescence signal is converted to a digital correlator ALV5000 / E (ALV G
(mbH). In addition, in practice, 20 μl of the sample droplet for measurement was spotted on a cover glass, and a small box was covered on the sample table so as to cover the cover glass in order to prevent evaporation during the measurement. The sample volume in the focal region was determined from the diffusion count of Rhodamine 6G. The volume element is composed of fluorescein and Flu-d
It was determined using the concentration of the UTP solution.

The fluorescence signal measured for about one minute was sequentially stored in a storage unit, and was evaluated by programming so as to be analyzed by applying it to a fluorescence autocorrelation function G (t). This fluorescence autocorrelation function G (t) is
Average number N of fluorescent molecules in the measurement region, free labeled substrate molecules (F) as mononucleosides (monomer DNA)
lu-11-dUTP) translation time taken incorporated the labeled substrate molecules as translational time tau _mono and polymeric DNA of tau
_{From poly} , the method of Rigler et al. (Fluorescence)
nce Spectroscopy-New Metho
ds and Applications, Spring
er, Berlin, 13-24, InJ. R. Lak
owicz (Ed.), 1992).

[0041]

(Equation 1)

[0042] In Equation 1, y is the proportion of the polymer DNA ^{_{components, τ mono = Wo 2 / 4D}} mono, τ poly = Wo 2/4
D _poly , S = Wo / Zo (where Wo is the diameter of a volume element of a substantially cylindrical measurement area (see FIG. 2B) formed in a minute visual field near the focal point, and 2Zo is its length ), D _mono and D _poly are the translational diffusion coefficients of monomeric and polymeric DNA, respectively.

The data analysis was further performed using a non-linear least squares parameter method to calculate the normalized mean square deviation between the data and the model. The total number T of molecules amplified through the desired PCR cycle is given by Equation 2; T = (N / V
c) By calculating with Vc, it was output as quantified data. Here, Vo is a volume element, and Vc is the volume of the solution collected by the spin column. FC
During S measurement, the average fluorescence intensity was also measured. The intensity reflects the total number of fluorophores (Flu-dUTP) incorporated into the amplified DNA in the volume element. The value obtained by dividing the measurement speed of the average fluorescence intensity by the number of DNA molecules Ny is DN
It was adopted as the average measurement speed C / M of the A molecule. This value could be used as an effective labeling concentration of the PCR product.

(3) Calculation of Measurement Area The volume element of the measurement area is a reference sample of rhodamine 6G, fluorescein (fluorescein).
n), and the measurement of the diffusion time of Flu-dUTP. The ratio between the length and the diameter in the measurement area (see FIG. 2B) provided near the focus was obtained from the value of S in Equation 1, and was about 5. The volume element V ₀ was calculated to be 1 × 10 ⁻¹⁵ l.

(4) Flu-11-dUT by PCR
Incorporation of P In order to clarify the effect of the fluorescently labeled nucleoside in PCR, the concentration of Flu-dUTP was changed from 10% to 50%. FIG. 3 is a transcript showing the characteristics of an amplification product of 4 kb in agarose electrophoresis. That is, 3 μl of a PCR reaction mixture as a test solution after 50 cycles of nucleic acid amplification reaction was applied to a 7% agarose gel. Here, FIG. 3 (A) shows the results before ethidium bromide staining and FIG.
(B) is after ethidium staining. In the figure, M
Indicates a molecular marker (HaeIII-digested phi X174 DNA). In the figure, “0”, “1”
"0", "20", "30", "40" and "50"
Indicates the% ratio between Flu-dUTP and dUTP + dTTP. FN is a fluorescent nucleoside (a non-incorporated nucleoside). The fluorescence emitted from the gel in FIG. 3A was measured using an orange optical filter Ya3, and the measurement in FIG. 3B was performed using a red filter R1.

According to FIG. 3A, 50% Flu-d
It can be seen that a 4000 bp fluorescent PCR product is synthesized even in the presence of UTP. The fluorescence intensity of the labeled DNA is
It increased with the concentration of Flu-dUTP. The total amount of amplified DNA can be shown by ethidium staining,
In this experiment, as shown in FIG.
It was shown to be the same as without u-dUTP.

(5) Fluorescence intensity and amplification product amount in PCR FIG. 4 is a graph showing the relationship between the label concentration and the product amount obtained as a function of the% ratio of Flu-dUTP in PCR. In the figure, square marks indicate the average measured value C / M per DNA molecule. Further, black circles indicate the number of DNA molecules in the measurement region which is the observation visual field. According to FIG. 4, it was found that the fluorescence intensity C / M per molecule increased with the increase of Flu-dUTP. This result indicated that the labeling concentration was dependent on the concentration of Flu-dUTP. And also, the number of DNA molecules was determined by the experimental Flu-d
It was almost constant within the UTP concentration range. This result was consistent with the gel electrophoresis analysis shown in FIG. This indicates that fluorescein does not interfere with the function of DNA synthase. Free Fl as monomer
Comparing each C / M value of u-dUTP and PCR product,
Flu-dU incorporated into a 4000 bp DNA strand
The number of TP monomers is known. It indicates that the modified nucleosides may be next to each other.

(6) Monitoring of PCR by FCS FIG. 5 is a graph showing the transition of the autocorrelation function in the course of the PCR reaction, and shows a typical time change of the correlation curve. In the figure, the circles indicate 10 cycles, the triangles indicate 20 cycles, the inverted triangles indicate 30 cycles, the diamonds indicate 40 cycles, and the squares indicate 50 cycles of the nucleic acid amplification reaction, respectively. As can be seen from FIG. 5, the value of the zero time delay (y-intercept) of the autocorrelation function is P
It decreases with the CR cycle. This result shows that DNA
This is due to the increase in the number of molecules.

FIG. 6 is a graph in which each autocorrelation function is normalized at a time delay of zero. In FIG. 6, the curve of Flu-dUTP is indicated by a black circle for comparison.
In the figure, the solid line is 10 cycles, the broken line is 20 cycles, the dotted line is 30 cycles, the dashed line is 40 cycles,
The dashed line is the value after each of the 50 cycles of nucleic acid amplification. As can be seen from FIG. 6, the autocorrelation function (solid line) obtained after 10 cycles is the Flu-dUT
There is a clear difference from that of P. Although this autocorrelation function could not be applied to a simple one-component model, it could be applied to a two-component model as shown in Equation 1 above. In addition, even in a test solution in which PCR was performed for 50 cycles, a short diffusion time component could be detected. The short components are unreacted Flu that has passed through the spin column.
-Defined as dUTP.

FIG. 7 shows P as a function of the number of PCR cycles.
4 is a graph showing the number of amplified DNA molecules in a CR reaction solution (5 μl). The total number of amplified DNA molecules was calculated according to Equation 2 above. Experimentally, Lambda Fage D
Various investigations were made on the initial amount of NA. In the figure, the square mark is 200p
g (3.75 × 10 ⁶ copies) of lambda phage DNA
Indicates an initial amount, and hereinafter, a circle indicates 20 pg.
(3.75 × 10 ⁵ copies), the triangle mark is 2 pg (3.7
5 × 10 ⁴ copies), the inverted triangle is 0.2 pg (3.75 copies)
× 10 ³ copies).

FIG. 8 shows that 20 as a function of the initial number of templates.
It is the graph which showed the number of DNA molecules after a cycle. DN
When A is amplified by PCR, the number of DNA molecules increases exponentially before the plateau effect is reached. The initial number of template molecules is 1024 (=
3.75 × 10 ⁶ copies (corresponding to 200 pg) of DNA result in 3.75 × 10 ⁹ copies because the DNA can be amplified by 2 ¹⁰ ) times. However, the initial copy number is 3.
At 75 × 10 ⁶ , after 10 cycles in FCS measurement 7.
44 × 10 ⁹ copies were measured. Similarly, when the initial number of copies was 3.75 × 10 ⁵ , 0.31 × 10 ⁵ copies were obtained after 10 cycles.
10 ⁹ copies were measured. Thus, the quantification of FCS measurements appears to be of low fidelity early in the amplification sample. This can be said to be a result of the distribution of a large amount of unincorporated labeled nucleosides in the detection region with a small amount of amplified DNA.

This direct label-based PC using FCS
In the R quantification, the initial concentration of the target DNA at the level of 10 ³ can be detected even after 10 cycles of amplification, but as can be seen from FIG. 8, the initial concentration of the target DNA can be better estimated after 20 cycles. In this figure, since the relationship between the initial number of DNA molecules and the number of products is linear, the number of target DNA molecules can be quantified by FCS measurement. Further, although the data is omitted, a similar relationship is obtained by using another curve 500b.
It was also confirmed that detection was possible with another DNA sequence of p. In addition, various experiments used 20 cycles of PCR, 5 μl of sample solution. It goes without saying that more precise and higher reproducibility can be obtained by increasing the amount of the sample solution. This is of paramount importance in diagnostic applications. Therefore, in order to save time and resources, it is preferable to appropriately select the number of cycles that can provide good reproducibility through experiments.

As shown in the above experimental results, in the present invention, FCS was used as a molecular counting method for direct label PCR.
Succeeded for the first time. In addition, 50% of dTTP was added to Flu-dU for a 4000 bp target nucleic acid.
It was proved that PCR was not inhibited by replacing with TP. For shorter target nucleic acids, eg, 500 bp, 1000 bp, 2000 bp, stronger fluorescent bands were detected by gel electrophoresis after 50 thermal cycles.
In this method, as a labeling reagent, a substrate molecule used for ordinary PCR, which is bound to a measurable label molecule such as fluorescence, is used, so that it can be easily used in an ordinary laboratory as in other ordinary PCR steps. Applicable to The FCS assay described here requires only minor changes in PCR conditions, as little as 5 μl of reaction solution as test solution, and a few PCR cycles. The fluorescently labeled molecule incorporated into the DNA molecule can be bound to any gene marker or infection marker, and while effectively utilizing the know-how of the established PCR method, the nucleic acid of various organisms can be used. Can amplify molecules.

FCS-based quantitative PCR according to the present invention
Is advantageous in many areas. This technique is sensitive, saves time and effort, and is very simple in the composition of the reactants and the procedure for their use. It should also be mentioned that the measurement of FCS is non-invasive, and that the test solution measured by FCS can be conveniently used for further experiments, for example, fluorescence hybridization and FISH analysis. Furthermore, FCS can also measure the restriction enzyme digestion process in a homogeneous solution. Therefore, as introduced here, densely labeled DNA
By using the method, analysis such as point mutation and RELF can be detected by FCS after amplification without using any separation method such as column chromatography or gel chromatography. These FCS
PCR-based quantification methods have been applied to diagnostics and screening experiments, and have opened up a new field of single molecule detection.

As described above, two important parameters in biological science can be obtained by FCS. It is the average number of molecules in the detection area (10 ^-15 l) and the translational diffusion coefficient of the molecules. In the present invention, PCR products were analyzed using FCS as a tool for single molecule measurement. P
Before the process of DNA amplification by CR reaches a plateau,
Quantification of a specific DNA base sequence can be performed. PCR
Fluorescein 11-dUT as a substrate for enzymatic amplification cycles to enable the application of FCS to
PCR in the presence of such labeled nucleosides
By performing 4000 bp of hail target DNA
Was able to be labeled. After 10 cycles, amplified DNA could be detected by increasing the diffusion time of the fluorescent molecule in the FCS detection region. The initial number of molecules of the target DNA in the volume (5 μl) of the small detection area for PCR detection ranged from 3.75 × 10 ⁶ copies to 3.7510 ³ copies.

A PCR quantification method based on fluorescence quenching, such as fluorescence energy transfer analysis, has been previously reported (Heid, CA et al., Genome Res.,
6, 986-994, 1996; Livak, K .;
J. , PCR MethodAppl. , 4,357-
362, 1995). Although this technique is commercially available, in addition to the first and second PCR primer pairs, two kinds of dyes are included in one probe sequence as in the NASBA and APEX methods described as the prior art. In. When designing the oligo base of the probe, not only consider the base sequence that does not compete with the base sequence of the primer,
It is also necessary to consider the melting temperature (Tm). In addition, the positions of the donor and acceptor dyes also affect the efficiency of increasing the fluorescence intensity and the 5 'nuclease activity of the polymerase. Another fluctuation spectroscopy (fluorescence cross-correlation spectroscopy) has been reported as a method for detecting a specific target gene in a homogeneous solution (Castro, A. et al., Anal.
Chem. , 69, 3915-3920, 1997).
Although the cross-correlation method can detect very low concentrations of the target sequence, it still requires two fluorescent probes in the experimental solution.

From the viewpoint of simplicity, PCS based on FCS
The CR quantification method has important advantages over the energy transfer method and the cross-correlation method. The reproducibility and sensitivity of PCR strongly depend on the type of enzyme (PolI type or type), primer sequence and reaction conditions. Once these conditions are determined, FA based on FCS
-PCR is readily adaptable to regular PCR assays with only minor changes in primer ratio. Therefore, routine PCR conditions can be easily changed to FA-PCR conditions. In addition, other amplification methods such as RT-PCR (reverse transcription PCR) can be changed to this method, and in the future, mRNA in one cell can be quantified.

In gel electrophoresis and column chromatography, PCR products are separated from primers and by-products.
However, the target product is diluted in the course of electrophoresis and extraction from the gel material, and has the opportunity to come into contact with nucleases and may denature. Conversely, FCS is convenient for sample collection after measurement, because sample droplets are simply placed on a cover glass, and because FCS is non-invasive, PCR solutions are not diluted, and There is no metamorphosis inside. High resolution of gel electrophoresis
Although the difference in base length can be detected, it usually takes about 1 hour for electrophoresis. On the other hand, FCS is a very high-speed measurement. Optionally, it may be longer than 1 minute, preferably 1 minute.
(For example, less than a minute), for example, there is an advantage in a large sample test such as a clinical test or a screening test. FCS
The sample after the inspection can be immediately analyzed in detail by gel electrophoresis or the like, and the FCS measurement result can be confirmed or the nucleotide sequence can be analyzed. And also, the collected sample can be used for subsequent experiments. For example, it can be used as a hybridization probe. If it is not necessary to collect the amplified sample after FCS measurement, it can be discarded without the risk of carryover contamination. This is because FCS is a non-contact measurement method.

[0059] Since FCS is non-invasive and allows direct measurement, the amount of PCR sample is determined by the amount of measurement required by the FCS. Although the present inventors measured this time with a sample volume of 10 μL, it could theoretically be reduced to a volume element level of 10 ⁻¹⁵ L. However, if the PCR volume can be reduced to 1 μL, compared to other methods,
This is a great economic advantage. Moreover, since FCS is based on a fluorescence microscope and the detection region can be placed on an objective lens, it can be used for quantification of specific RNA or DNA by in-situ PCR.

The inventors have obtained good results by using a two-component model for the analysis of the PCR solution. However, in other experiments many DNA species may be present. In such cases, the DNA species can be well separated by conventional gel electrophoresis. Differences in the correlation curves from the model functions of the observed FCS data can suggest the presence of other components distributed in the solution, but a simple two-component model would have limitations in such cases. . As a result, if a more detailed quantitative analysis is needed, CONTIVN
It is preferable to develop a multi-component FCS analysis method to obtain a distribution function by slightly revising a computer program such as (from Max-Plant Institute) to follow the method of the present invention described above.

In the measurement step in the present invention, noninvasive and noncontact measurement can be performed without using physical separation means, and the amount of sample used is small. Relatively low PCR
When analyzing under measurement conditions where the initial amount of the target nucleic acid molecule is clearly small, such as when measuring by the number of cycles, prior to the measurement step, the labeled molecule that has not been incorporated in the amplification process is subjected to gel filtration or the like. Appropriate Bound / Free
If the above-mentioned non-invasive and non-contact measurement is performed after removal by the separating means, there is also an advantageous aspect that the S / N ratio can be easily increased and the sensitivity can be increased. Physical handling of such small samples can be accomplished using capillaries, thin coverslips, or small holes on the film-covered glass surface, and can be used for real-time analysis. With these properties and further improvements it is clear that monitoring of the amplification process is easy (real-time monitoring) and that an automatic detection system (robotics) is also possible. We are FA based on FCS
-Conclude that the PCR quantification method has advantages in sensitivity, quantification accuracy and simplicity. This method is expected to open a new era of molecular diagnostics as well as rapid screening.

[0062]

According to the present invention, the amplification reaction by PCR is performed by labeling the substrate molecule with the label molecule, and the mobility of the label molecule is measured. Can be accurately and quantitatively detected with a simple configuration.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing an example of an apparatus for performing the method of the present invention.

FIG. 2 is a partially enlarged view of the apparatus of FIG. 1;

FIG. 3 is a transcript showing characteristics of an amplification product of 4 kb in agarose electrophoresis.

FIG. 4 is a graph showing the relationship between label concentration and product amount obtained as a function of the Flu-dUTP% ratio in PCR.

FIG. 5 is a graph showing a transition of an autocorrelation function in the course of a PCR reaction.

FIG. 6 is a graph in which each autocorrelation function is normalized at a time delay of zero.

FIG. 7 shows PCR as a function of PCR cycle number.
7 is a graph showing the number of amplified DNA molecules in a reaction solution (5 μl).

FIG. 8 is a graph showing the number of DNA molecules after 20 cycles as a function of the initial number of templates.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Fluorescence microscope, 2 ... Photomultiplier, 3 ... Data processing device, 4 ... Display device, 11 ... Sample containing liquid, 12 ... Sample stand, 13 ... Slide glass, 14 ... Lid, 15 ... Objective lens, 16 ... Laser Generator.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA35 DA12 DA13 DA14 FA11 FA16 FB01 FB07 JA01 4B024 AA01 AA11 AA20 BA80 CA01 HA08 HA11 HA14 HA17 4B029 AA07 BB13 FA01 4B063 QA01 QA19 QQ10 QQ42 QR08 QR42 QR58 QR62 QR58 QR58

Claims (17)

[Claims] 1. A method for analyzing a target nucleic acid utilizing a nucleic acid amplification reaction by a test solution containing a primer, a substrate molecule, a nucleic acid synthetase, and a target nucleic acid molecule, wherein the method is labeled with a label molecule capable of generating a measurable signal. Performing a nucleic acid amplification reaction with a test solution containing the substrate molecules obtained, a step of measuring a signal from the labeled molecule for the nucleic acid-amplified test solution, and a step of measuring a signal from the labeled molecule based on the measured signal. A method for analyzing a target nucleic acid, comprising the steps of: evaluating the ease of movement of the target nucleic acid; and quantifying the target nucleic acid molecule based on the evaluation result. 2. The method according to claim 1, wherein the measuring step includes a step of measuring the abundance of the labeled molecule in a predetermined measurement region. 3. The method according to claim 2, wherein the measuring step measures the movement amount within a predetermined time a plurality of times. 4. The method according to claim 3, wherein the evaluation step includes a step of converting the measured data into statistical data representing a change in the movement amount. 5. The method of claim 4, wherein converting comprises performing an operation with an autocorrelation function. 6. The method according to claim 6, further comprising a step of removing the labeled substrate molecule not involved in the nucleic acid amplification reaction between the step of performing the amplification reaction and the measuring step, wherein the measuring step is performed without contacting the test solution. The method according to any one of claims 1 to 5, wherein the measurement is performed. 7. The method according to claim 1, wherein the quantifying step includes a step of judging the presence or absence of a labeled molecule incorporated during nucleic acid amplification based on the evaluation result. . 8. The method according to claim 1, wherein the quantifying step includes a step of determining the number of target nucleic acid molecules incorporated during the nucleic acid amplification based on the evaluation result. Method. 9. An apparatus for analyzing a target nucleic acid utilizing a nucleic acid amplification reaction by a test solution containing a primer, a substrate molecule, a nucleic acid synthetase, and a target nucleic acid molecule, wherein the target nucleic acid is labeled with a label molecule capable of generating a measurable signal. Holding means for holding a test solution containing the obtained substrate molecule, measuring means for measuring the signal from the labeled molecule after the nucleic acid amplification reaction with respect to the held test solution, and measuring the signal from the labeled molecule based on the measured signal. A target nucleic acid analyzer, comprising: an evaluation unit for evaluating the ease of movement of the target nucleic acid; and a data output unit for outputting data obtained by quantifying the target nucleic acid molecule based on the evaluation result. 10. Apparatus according to claim 9, wherein the measuring means comprises an optical system for performing the measurement in a microscopic field provided in a diffraction limited area near the focal point. 11. The apparatus according to claim 9, wherein the measuring means includes a microscope for performing a measurement in a microscopic field formed by the confocal optical system. 12. The diffraction limited area has an average diameter of 30 ± 20 μm.
Device according to claim 10 or 11, characterized in that it is formed by m apertures. 13. The diffraction limited area has an average diameter of 20 ± 10 μm.
Device according to claim 10 or 11, characterized in that it is formed by m apertures. 14. A microscopic field having an average radius of 200 ± 50 nm.
12. The device according to claim 10, wherein the device is a substantially cylindrical region having an average length of 2000 ± 500 nm on the optical axis. 15. An evaluation means comprising: means for storing a plurality of measurement data obtained within a predetermined time; and calculation means for calculating the stored plurality of measurement data by an autocorrelation function. Device according to claim 9. 16. An evaluation means includes means for storing measurement data on a plurality of label molecules obtained in a measurement area, and calculation means for calculating the stored measurement data for each label molecule using an autocorrelation function. 16. The method according to claim 15, wherein
An apparatus according to claim 1. 17. The data output means includes conversion means for converting, based on a result calculated by an autocorrelation function, to statistical data representing a temporal change in a plurality of tracking data. Apparatus according to claim 15 or claim 16.