JP3387707B2 - Gene analysis method and apparatus - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and apparatus for analyzing genetic information in the fields of medical diagnosis and life science.

[0002]

2. Description of the Related Art In recent years, with the progress of gene analysis technology,
In the fields of medical diagnosis and life science, analysis of genetic information has become important. For example, in the field of life sciences,
As typified by the Human Genome Project, projects to determine the entire nucleotide sequences of various animal and plant genes are in progress, and coding regions for novel proteins, regulatory regions for expression, and related genes such as oncogenes Is gradually being revealed.

On the other hand, in the field of medical diagnosis, in response to these research results, a movement to introduce gene analysis technology into various etiological diagnosis and tests has started. Hepatitis C and HIV (Human
Immunodeficiency virus (AIDS) infection is the most common diagnostic method for infectious diseases such as AIDS (acquired immune deficiency syndrome) because of the high sensitivity of detection and the relationship between infectious characteristics of these viruses (retrovirus) and polymorphism This is an example of a field in which the introduction of genetic diagnosis is strongly desired. In addition, gene analysis that provides accurate and detailed information for tumor cell tests, which currently rely on empirical pathological diagnosis, and HLA (human leukocyte antigen) type tests, where the number of specimens is rapidly increasing due to the demand for bone marrow bank registration. It is hoped that the technology will be introduced.

Recent advances in gene analysis techniques desired in such fields are remarkable, and in addition to the conventional typical nucleotide sequence determination method and probe method, slight sequence differences in genes are electrophoresed. A method has been developed for separating by utilizing the difference in the higher-order structure of single-stranded DNA. This method is based on SSCP (Single Strand Conformation Polymorphism
s), for example Genomics Vol. 5, 874
Pages-879, 1989 (Genomics Vol.5, pp874-pp87
9,1989), it has attracted attention as a method that can detect even single nucleotide substitution with high sensitivity. In this separation method, when a complementary DNA consisting of a pair of double-stranded DNAs is placed in a single-stranded state, the single-stranded DNA is usually intramolecularly produced under appropriate conditions (ionic strength, temperature, etc.). By self-associating to form a sequence-specific higher-order structure, we focused on the fact that different higher-order structures differ when the sequences differ, and by detecting this difference in higher-order structure as a difference in mobility by gel electrophoresis. It detects a sequence difference.

This method has high separation sensitivity for sequence differences, but since electrophoresis is used as a separation method, it requires a long time for separation and it is difficult to achieve high throughput.
It is difficult to select conditions for efficiently reflecting the difference in sequence in the difference in electrophoretic mobility. Further, it is not only difficult to automate, but in some cases, complete separation of all polymorphisms requires separation under a plurality of conditions.

[0006] Similarly, a method called denaturing gradient gel electrophoresis has been proposed as a method for detecting a slight sequence difference in a gene (for example, SG Fischer.
and L.S.Lerman: Proc.Natl.Acad.Sci.USA, Vol.80, 1579
-1583, 1983), which also uses electrophoresis for the separation method, and has the same drawbacks as the above-mentioned problems.

On the other hand, a double-stranded D having a completely complementary sequence
The difference in denaturing conditions between NA and double-stranded DNA that has an incomplete but almost complementary sequence is
Difference in absorbance change when denaturing to NA (denaturation curve (me
lting curve)) (for example, IV Razlutuskii, LS Shlyakhtenko and Yu.
L. Lyubchenko: Nucleic Acids Research, Vol.15, No.
16, p6665-6676 (1987)). Furthermore, the type of single-stranded RNA having a higher-order structure (hairpin, stem, loop structure, etc.) can be identified as a change in absorbance (denaturation curve) when the base pair of single-stranded RNA is separated. It has also been known (for example, LG Laing and DE Draper: J. Mol.
Biol. (1994) 237, 560-576).

[0008] According to these techniques, the operation of electrophoresis is not required after the sample is obtained, so that the operation is simple and the measurement itself is easy to handle as an optical measurement method and is reliable. It has the advantage that it is highly reliable.

Further, a double-stranded D having a completely complementary sequence
Optically measuring the phenomenon in which double-stranded DNA having a sequence that is almost completely complementary to NA is resolved by fluorescence energy transfer caused by two types of fluorophores modified in each complementary strand upon denaturation There has been proposed a technique for detecting a sequence difference between two types of DNA which are not completely complementary by the detection by the method (Japanese Patent Application Laid-Open No. 7-31500). In this case as well, there is no need for the operation of electrophoresis after the sample is obtained, and there are the same merits as the above-mentioned example.

[0010]

However, what can be detected by these methods is, for example, the sequence composition (GC content etc.) and the deletion / insertion of bases, and detailed sequence differences, especially single base substitution, can be detected. The genotyping involved was virtually difficult. In addition, control of denaturing conditions must be performed at an extremely low speed, making it difficult to adapt to high-throughput detection and determination.

Further, it has not been considered to directly analyze the genotype containing single nucleotide substitution directly from single-stranded DNA.

In order to solve this problem, the invention of the present application directly detects a denaturation curve (melting curve) of the higher-order structure of single-stranded DNA, which simplifies the apparatus structure and provides a more accurate and realistic signal. A processing method and apparatus are provided.

[0013]

According to the present invention, single-stranded DNA of a target gene region can be separated and analyzed even in the case of a heterozygous type having two or more genotypes at the same time. A denaturation curve corresponding to all known polymorphisms is stored (template curve), all combinations of curves created by linear combination of one or more template curves,
A single-stranded DNA fragment (sample) in which the combination of the template curves was measured when the mean squared error was less than or equal to a predetermined value and was the smallest when the signal curve obtained from the sample was compared.
It was configured to recognize as the type of gene, that is, the sequence characteristic.

Further, when considering the gene analysis in the medical diagnosis applying the PCR method, what is to be acquired as the gene information is the presence / absence of amplification of the target gene fragment and the sequence information of the target gene fragment when amplified. In the present invention, the melting curve
It has been devised to be advantageous in terms of obtaining at the same time by quantitatively measuring.

Further, by devising the sample holding means for holding the sample and controlling the temperature to have a large surface area / volume ratio, the denaturation rate can be increased, and the result of the measurement is as follows.
It was discovered that the denaturation curve of single-stranded DNA shows a different curve when the temperature rises and the higher-order structure is solved, and when the temperature lowers and the higher-order structure is formed, showing hysteresis. The hysteresis curve itself has reproducibility, and the genotype of single nucleotide substitution could be analyzed by processing this data by the same signal processing means as above.

Furthermore, when preparing a single-stranded DNA sample,
Excitation is achieved by incorporating an intercalator such as ethidium bromide that intercalates into the base pairing formed by the complementary sequence of DNA to shift the fluorescence wavelength, and irradiates the sample solution with excitation light of predetermined wavelength. It was discovered that the fluorescence generated by the irradiation of light causes a change in the fluorescence intensity generated by the interaction between the single-stranded DNA fragment sample and the intercalator when the single-stranded DNA is denatured. Therefore, the sequence information of the single-stranded DNA fragment can be obtained by measuring this and processing this data by the signal processing means similar to the above.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION One of the embodiments of the present invention will be shown below.

FIG. 1 is a block diagram showing the basic configuration of the gene analysis apparatus of the present invention. The ultraviolet light (wavelength 260 nm) emitted from the light source 1 is split into two by the optical system 2 and is incident on the sample section 4 and the control section 5 installed in the sample chamber 6. After that, each light beam collected by the optical system (not shown) is detected by the photoelectric converter 7 and analyzed by the amplifier 9 and processed by the signal processing device 10. The sample chamber 6 can control the temperature of the sample part 4 and the control part 5 according to a temperature profile arbitrarily programmed by the temperature controller 8. The temperature control can be performed at an arbitrary temperature raising / lowering rate. The sample temperature in the sample chamber 6 is measured by a temperature sensor (not shown), fed back to the temperature controller 8 and input to the signal processing device 10. Sample temperature is -20 ℃ to 100 ℃ depending on the sample
Therefore, when the sample unit 4 is controlled to a temperature equal to or lower than room temperature, dew condensation should not occur on the surface of the sample chamber 6 or the cell holding the sample (sample cell).
The structure is such that dry air flows from the lower part of the sample chamber 6 upward.

The control section 5 is provided to correct the absorption by the buffer solution or sample cell in which the sample of the sample section 4 is dissolved and to obtain the net absorption by DNA. this is,
This is a conventional method in the spectroscopic measurement technique, and all the data of the examples described below are corrected in this way.

Next, an example of gene polymorphism analysis using the above apparatus will be described.

In this example, the HLA (human leukocyte antigen) class 2: DQA1 region was subjected to asymmetric PCR amplification from genomic DNA extracted from human blood cells, and gene polymorphism analysis (typing) was performed on the region.

DNA obtained by extracting genomic DNA by a conventional method
Sample solution, final concentration 10 mM Tris-Hcl (pH 8.3), 50 mM Kcl, 1.
5mM MgC12, 0.02% gelatin, 200μM each containing deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP)
PCR buffer, 2.5U Taq polymerase, two types of primer GH corresponding to the DQA1 region of HLA to be analyzed
26 and GH27 (; Ulf B. Gyllensten and Henry AE
rlich Proceedings National Academy of Sciences You. S. A (Proceeding Nation
al Academy of Science of USA) Vol.85, pp.7652-765
6, October 1988) 20 pmol each was mixed in a tube, mineral oil was filled in the upper layer, and then PCR was performed. P
Parameter of CR cycle is 94 ℃ (1 minute) → 57 ℃
(1.2 minutes) → 72 ° C. (1 minute), 27 cycles.

Using this reaction product 1/100, asymmetric P
Perform CR. The asymmetric PCR reaction solution is almost the same as the above PCR, only 10 pmol of GH26 and GH27 of primer are used.
Change the amount to 1pmol. The parameters of the PCR cycle are 94 ° C (1 minute) → 57 ° C (1.2 minutes) → 72 ° C.
(1 minute) is 15 cycles.

This reaction product was microfiltered: microc
on (TM) 30 (Grace Japan Co., Ltd.) for desalination and concentration TNE
Buffer l (10mM Tris-Hcl, 1mM EDTA (pH8.0), 30mM Na
cl) and used as a sample solution.

Incidentally, although asymmetric PCR was used for the preparation of single-stranded DNA in this example, double-stranded DN was used as another method.
Method in which A is PCR-amplified and then one-sided single strand is digested with λ exonuclease (λ exonuclease method)
Alternatively, PCR is performed with one of the primers used for PCR being immobilized on the membrane, and then the single-stranded D that is not immobilized on the membrane with the temperature raised to the denaturation temperature.
A method of washing out NA (membrane method) can be adopted. The membrane method requires an operation of immobilizing a predetermined primer on the membrane, but it is possible to prepare a single strand with high purity in a very simple manner and is suitable for automation.

The above sample solution was placed in the sample section 4, and the TNE buffer 1 as a control solution was placed in the control section 5, and once the sample temperature was lowered to 0 ° C., the temperature was raised to 1 ° C./min at 60 ° C. The temperature was raised to.

2 to 7 (a) and (b) show the HLA-DQA1 region gene (242 bases or 23 bases by the above method).
6 known types out of 9 bases (0101, 0102, 0103, 0301,
0401, 0601: The WHO nomenclature committee for facto
rs of the HLA system, 1989.Immunogenetics, 31: 131-14
(1990, 1990) denaturation curve (melting curve)
curve) is shown as absorbance data and differential absorbance data obtained by differentiating this with temperature (other 0201, 050
Regarding the two types of 1), since the inventor could not obtain a homozygous sample, it was omitted here). Also (b)
Shows only the differential absorbance data. Where 0101 and 0102
Is 1 base difference, 0101 and 0103 are 3 bases, and 0102 and 0103 are 2 bases. 0101 and 0301 differ by 27 bases, 0401 and 0601
Is 3 bases shorter than the other types, and the sequence differs by 20 bases or more compared to 0101, but 0401 and 0601 differ by only 1 base.

Further, although (a) and (b) show the analysis results for the same sample, (a) shows the analysis results obtained at the time of filing the priority application of the present application, and (b). Is the analysis result obtained relatively recently. Despite the fact that the two show the analysis results for the same sample, the inconsistency is due to the difference in the maturity of the analysis technique and the correction of the data adopted to compensate for the immaturity of the analysis technique. This does not mean that the analysis is not reproducible.

As shown in the figure, first, groups having large arrangements, for example, 0101 to 0103 (type 1), 0301 (type 3), and 0401.
It can be seen that the melting curve features are clearly different from those of 0601 (short type). In addition, there are also differences in types that are 1 base difference or 2 base differences (for example, 0101 to 0103 or 0401,
Although it is not the main peak, it can be detected as an obvious difference.

FIG. 8 shows a melting curve of 0301 type and its differential function approximated by superposition of Gaussian distribution functions. As is clear from FIG. 8, type 0301 meltin
The g curve is well approximated by the superposition of two Gaussian curves.

This means that if the denaturation curves (melting curves) of these known polymorphic DNAs are examined and the sample DNAs are compared using the denaturation curves (melting curves) of these known polymorphic DNAs as templates. Means that the sample DNA can be specified. Furthermore, for heterozygous DNA, the sample DNA can be specified by analyzing the Gaussian curve and then specifying the overlap.

Table 1 shows a: size, μ: peak when differential functions of six known melting curves shown in FIGS. 2 to 7 are similarly expressed by plural kinds of Gaussian curves. Position (average value), σ: extent of spread (standard deviation).

[0033]

[Table 1]

The number of terms of the Gaussian function used for the approximation is the point where the approximation error is saturated to the minimum value. As shown in this table, it can be seen that the differential function of the melting curve corresponding to each type is represented by characteristic parameters (a, μ, σ). That is, when comparing the denaturation curve (melting curve) of these known polymorphic DNAs with an unknown gene (type) as a template, if it is carried out in the form of comparison with the parameters shown in Table 1, automatic calculation processing is performed. Not only can it be performed as, but also a strict comparison can be made by mathematical processing.

As the mathematical processing, all combinations of curve data created by linear combination of one or more template curve data of known denaturation curve data prepared in advance, and the signal newly measured and input. By comparing the curve data with the template curve data having the least statistical error, or the combination of the template curve data which is the basis of the curve data generated by the linear combination thereof, a combination prepared from the sample double-stranded DNA fragment was prepared. The sequence characteristic of the double-stranded DNA fragment may be used.

FIG. 9 shows the derivative function of the melting curve and the characteristic parameters of the curve data for the heterozygous samples 0102 and 0301. Even in the case of heterozygous type, it is expressed by superimposing each gene type, and it is understood that typing can be performed by the capacity of spectrum analysis.

Table 2 summarizes the parameters for each genotype from the results shown in FIG. That is, when a heterozygous gene is represented by Gaussian curve data, if parameters μ: peak position (mean value) and σ: spread degree (standard deviation) show almost the same values as the regression values in Table 2, then It can be identified as a heterozygous type of the two suspected genotypes.

[0038]

[Table 2]

In this embodiment, the maximum is 5 ° C./min.
Sufficiently accurate denaturing curve data (melting curve data) in a practical sense was obtained at the heating rate of. In this case, the analysis time is about 10 minutes for the analysis of one sample, which is 20 times faster than the conventional sequencing or SSCP method (4 hours or more).

FIG. 10 shows another analysis example based on FIG. 2 (b) -FIG. 7 (b) for heterozygous DNA. Also in this example, the exon2 region of HLA-DQA1 (24
For 8 types of polymorphisms with 2 bases or 239 bases (3 bases deleted), denaturing curve data (melting curve data) serving as a template for each sense strand is measured in advance. The heterojunction type actual measurement curve data obtained from the sample shows DQA1 * 0101 Template 0101 and DQA1 * 010
It is shown that they are in good agreement (mean squared error RMS = 0.00008) as a synthetic curve of template 0102 showing 2.
It shows that the sample can be judged to be a heterozygous DNA of both.

In polymorphism analysis, it is necessary to determine which of the known genotypes the genotype of the sample to be analyzed belongs to, or whether the genotype is novel. It is also necessary to be able to perform separation / analysis of heterozygotes having multiple genotypes simultaneously, but in this analysis example, separation / analysis of heterozygotes simultaneously having multiple genotypes is facilitated. As signal processing, denaturation curve data corresponding to all known polymorphisms are stored (template curve data), and all combinations of curve data created by linear combination of one or more template curve data The measured and input signal curve data is compared, and when the mean square error is less than or equal to a predetermined value and is the smallest, the combination of the template curve data is measured for the gene type of the single-stranded DNA fragment ( That is, the genotype can be determined easily and more reliably by recognizing it as a sequence feature). It is also possible to output as probable combinations in the ascending order of the mean square error.

With respect to the results shown in FIG. 10, Table 3 explains the effectiveness of the combination that gives the smallest error.

As a result, the combination giving the smallest error shows the correct heterojunction type combination, and the error amount is
It was about the same as (but smaller than) the reproduction error obtained by measuring the melting curve data 5 times. Table 3 shows the mean squared error of the reproduction error and the correct combination (combination indicated by double circles in the table), and the mean squared error of some of the other combinations with a small error. Show. In the case of heterozygosity due to the combination of sequences with single nucleotide differences, the genotype that is most likely to be misjudged is the homozygous type due to each genotype of the groups that make up the heterozygote. It can be seen that there is a significant difference between the correct and incorrect answers.

[0044]

[Table 3]

Further, by controlling the temperature of the sample at a high speed and substantially uniformly, in some samples, when the temperature is controlled at a rate of temperature increase and temperature decrease exceeding 10 ° C./min, the single-stranded DNA becomes high. The dynamic temperature characteristics of the secondary structure could be measured. That is, during melting of higher-order structure (temperature increase) and during formation of higher-order structure (temperature decrease)
And the hysteresis curve (1 → 2 → 3 → 4 → 5 → 6 → 2 →
It was measured to draw 3 → 4 → 5 → ...). It was also found that these hysteresis curves differed depending on the specimen sample and were specific due to the difference in sequence due to base substitution, deletion or insertion. This is not limited to the change in absorbance described above, by a method similar to the signal processing method for the hysteresis curve of the absorbance, by evaluating the match between the hysteresis curve to be the template and the measured hysteresis curve, it is possible to perform faster and more accurate measurement. This means that it is possible to make various typings.

In the case of this method, it was possible to obtain the hysteresis curve of temperature rising / falling 2 cycles within 1 minute (50 seconds) per sample at the maximum and complete the measurement. The hysteresis curve changes according to the temperature rising rate. Therefore, if the heating rate is set to an appropriate value according to the polymorphism, a hysteresis curve corresponding to the genotype can be effectively obtained, and thus the gene analysis based on the heating rate dependency of this hysteresis curve can also be performed.

Next, an example of a gene analysis apparatus which performs continuous processing from pretreatment to measurement of a melting curve as a flow system using PCR as pretreatment will be described with reference to FIGS. 12 and 13.

FIG. 12 is a schematic flow chart of the process, FIG. 13 is a schematic diagram of the apparatus configuration, and FIG. 14 is a detailed configuration diagram of the spectroscopic cell.

The reaction process proceeds after passing through the steps (a) to (g) shown in FIG. As shown in (a), PCR is carried out in a PCR reaction cell 600 in which an oligonucleotide A602 serving as a PCR primer is immobilized on a porous filter film 601 on the bottom surface inside the PCR reaction cell. The PCR reaction cell 600 provided with the extracted and purified genomic DNA as a sample is shown in FIG.
Assemble to the device of 3. As the PCR reaction proceeds, as shown in (b), the double-stranded DNA (P) corresponding to the sample DNA is formed in such a manner that one end of the solid phase is fixed to the filter film 601 at one end.
CR product) is produced. When this double-stranded DNA is denatured, as shown in (c), single-stranded DNA (solid phase) with one end fixed to the filter film 601 and free 1-stranded DNA as shown in (d) are obtained. Separated into single-stranded DNA (liquid phase). This liquid-phase single-stranded DNA is dissolved in a measurement buffer solution, sent to a spectroscopic cell, the temperature of the spectroscopic cell is controlled, and the (melted) liquid-phase single-stranded DNA shown in (e),
The formation of the higher-order structure shown in (f) is controlled. With this spectroscopic cell, the absorbance can be measured to obtain a denaturing curve (melting curve) as shown in (g).

FIG. 13 shows a schematic diagram of the apparatus configuration. P through the inlet / outlet ports 705 and 706 of the sample pretreatment cell 700
As will be described later, a PCR reaction solution, a washing buffer solution, and a spectroscopy buffer solution are sent to the CR reaction cell 600, and the PCR reaction cell 600 is controlled by a predetermined thermal cycle. Primer A is used in the PCR reaction cell 600.
Is provided with a porous filter 601. In the sample pretreatment cell 700, (a)-(d) described in FIG.
Will be performed.

Oligonucleotide A serving as a PCR primer is added to the porous filter 601 in the PCR reaction cell 600.
Is immobilized at 10 pmol, and 100 ng of the extracted and purified genomic DNA as a sample is supplied to the PCR reaction cell 600. P
A gas source 707 and valves 723 and 7 are provided in the CR reaction liquid tank 701.
Gas is sent in via 22 and 721, and 50 μl of the PCR reaction solution is sent into the PCR reaction cell 600 via valves 725 and 724. 50 mM as PCR reaction solution
Kcl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgC12,
In a buffer solution of 0.001% gelatin (final concentration),
10 pmol each of primer (oligonucleotide) B and 10
nmol deoxyribonucleotide triphosphate (dNTP: d
ATP, dCTP, dGTP, dTTP) and thermostable DNA polymerase (Taq polymerase) mixed in 1 unit were used.

In this state, PCR is performed by controlling the temperature of the PCR reaction cell 600 with hot air or cold air. The number of cycles is 25, and the temperature condition is 94 ° C 30
Seconds, 55 ° C for 1 minute, 72 ° C for 30 seconds, total reaction time is 5
It was 0 minutes. It is considered that the time required for this cycle can be shortened to about 20 minutes by making the shape of the cell more elongated and increasing the area / volume ratio. The evaporation of the reaction solution at high temperature is prevented by the valves 724 and 72.
By closing 8 it was possible to make it virtually zero.

After the reaction, gas is supplied from the gas source 707 to the cleaning or spectroscopic buffer solution tank through the valves 722 and 723, and the cleaning buffer solution is flown to the filter 601 through the valves 726, 725 and 724. , Filter 6
Residual primer and residual dNTP by washing 01 several times
To remove. Waste liquid is passed through the valve 728 to the waste liquid tank 7
Abandoned in 03. The PCR reaction product remains on the filter 601. When there is a possibility that undesired substances that do not pass through the filter 601 may remain, the gas may be sent from the gas source 708 through the valve 728 and the waste liquid may be discarded into the waste liquid tank 709 through the valve 724. All of these operations are performed under the condition that the double strand is in the solid phase state on the porous filter 601 as shown in (b) of FIG.

After that, the last washing solution (which serves as a spectroscopic buffer solution) is introduced into the PCR cell 600, and then the temperature in the PCR reaction cell 600 is raised to a denatured state, and the solution is used as a gas source 708. Then, it flows from the PCR reaction cell 600 toward the entrance / exit 705. That is, as shown in FIG. 12D, the single-stranded DNA in the liquid phase is taken out, and the spectroscopic cell 300
Send it to. In this example, 20 μl of TNE buffer (10 mM Tris-HC was used as a washing buffer.
1, 1 mM EDTA (pH 8.0), 30 mM NaCl) was used. The sample introduced into the spectroscopic cell 300 is measured,
The washing buffer is flown through the valves 726 and 727 to be discarded in the waste liquid tank 704. As a result, the used sample is removed and the inside of the spectroscopic cell 300 is cleaned. At this time, it is possible to install a fraction collector instead of the waste liquid tank 704 and collect the sample after measurement.

In the above apparatus configuration, the number of PCR reaction cells 600 and spectroscopic cells 300 was one to one, but a plurality of PCR reaction cells were combined into one spectroscopic cell through a valve, and reaction products were sequentially spectroscopically separated. The measurement may be performed by being introduced into the cell.

Further, although the overall configuration of the system may be slightly complicated, a living cell sample such as blood, which is the source of the sample, is directly supplied to the PCR reaction cell 600, and a sample is prepared as a sample by a known method. It is also possible to make the system from extraction to analysis consistent by performing the process of extracting the gene of 1) and then performing the above-described genotype analysis process.

FIG. 14 shows the spectroscopic cell 30 adopted in FIG.
It is a figure which shows the structural example of 0, a side view is on the left side,
The cross-sectional view which saw the side view in the position of AA in the arrow direction is shown.

The spectroscopic microcell 300 of this embodiment is
A quartz glass box and a black quartz glass box, in which the optical path window is made of quartz (transparent) and the upper part of the rectangular parallelepiped is open, is formed. The sample inflow port 303 is left on both sides inside this upper part. A spacer made of black quartz glass is provided. Therefore, a square sample solution holding portion having an optical path length of 10 mm and an optical path cross section of 1.4 mm × 1.4 mm is formed below the spacer. At the center of the spacer, the temperature sensor 301 is slightly projected toward the sample solution holding portion. The spectroscopic microcell 300 is arranged in a temperature controller 302 for controlling the temperature of the sample solution in a built-in manner as indicated by a thick arrow (inserted) in the drawing.

The cell wall is made of black quartz glass in the sense that the reflected stray light is extremely small and the thermal conductivity and the strength are relatively high, and the thickness thereof is set to about 1 mm. Is suitable, and as another example, an aluminum alloy coated with platinum black or TiN (titanium nitride) may be used. In the cell of this embodiment, the temperature of the sample is measured by the temperature sensor 301 at the center position of the optical path and is embedded in the temperature controller 302. Therefore, the temperature of the sample is controlled with high efficiency,
Moreover, the temperature of the sample can be measured accurately. Further, the cell of the present example has a large surface area / volume ratio of 2800 as compared with a commercially available general spectroscopic cell, and this cell can raise and lower the temperature at a rate of 0.1 ° C./min at 5 ° C./sec. It has become possible.

Finally, an example in which denaturation curve (Melting Curve) was obtained by detecting fluorescence of sample DNA and intercalator using ethidium bromide will be described.

FIG. 15 is a block diagram showing an embodiment of the structure of a detection device for detecting the fluorescence of DNA and intercalator. Ultraviolet light emitted from a light source 801 (wavelength 260
(nm) passes through an optical system 802 such as a filter and a lens and enters a sample 804 installed in a sample chamber 805. Sample D
The presence of ethidium bromide intercalated in NA causes fluorescence at 590 nm, which is followed by optical system 807.
The fluorescence collected by the light is detected by the photoelectric converter 808,
It is processed by the analysis and signal processing means 810 through the amplifier 809. The sample chamber 805 can control the sample temperature according to a temperature profile arbitrarily programmed by the temperature controller 806. The temperature control can be arbitrarily set from a temperature raising / lowering rate of 0.1 ° C / min to a rate of 2 ° C / sec.
It can be controlled from 20 ° C to 100 ° C. Also in this embodiment, the spectroscopic cell shown in FIG. 14 can be used, and it is the same as the previous embodiment that the signal is handled and necessary measures are taken so that dew condensation does not occur on the surface of the cell.

Denatured curve (Me for fluorescence of intercalator)
In the case of lting curve measurement, the fluorescence intensity decreases as the single-stranded DNA higher-order structure melts. This is because the fluorescence emitted by the intercalation of ethidium bromide with the base pairs forming the higher-order structure disappears as the higher-order structure melts and the intercalation disappears. Initially, there was a problem of reproducibility such as change in fluorescence intensity depending on the concentration of ethidium bromide, but the denaturation curve (Melting Curv) standardized based on the fluorescence intensity at the lowest temperature was used.
By using e), it became possible to guarantee reproducibility among the same samples.

FIG. 16 is a fluorescence denaturation curve (Melting Cur) of HLA-DQA1 * 0101 and DQA1 * 0102.
ve) is shown. As in the case of using the absorbance, it was possible to separate single base substitution. Further, the data in FIG. 16 is measured at a concentration of 1/50 when the absorbance is used. By using fluorescence, sensitivity improvement of 1 to 2 digits (measurement accuracy of denaturation curve = improvement of S / N ratio) was realized.

The signal processing is the same as in the case of the above-mentioned absorbance.
As a result of comparison with the template curve, accurate analysis was possible for all the samples performed.

Although some examples of the present invention have been described above, the scope of application of the present invention is not limited to these examples, and the denaturation curve (Melti) of single-stranded DNA is not limited thereto.
It is within the scope of the present invention to provide a method and device characterized by obtaining information on the sequence by analyzing the ng curve).

[0066]

As described in detail above, by using the apparatus and method of the present invention, at least the minimum genetic information necessary for medical diagnosis and genetic testing, that is, presence / absence of the target sequence, abundance and sequence, is present. It was possible to obtain the characteristics, and the whole process from pretreatment to acquisition and analysis of genetic information could be realized in a short time with a simple device configuration and operation.

[Brief description of drawings]

FIG. 1 is a block diagram showing the configuration of a detection device according to a first embodiment of the present invention.

FIG. 2 (a) and (b) are HLA-DQA1 region genes.
The figure which showed the denaturation curve (melting curve) obtained about the known 0101 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

3 (a) and 3 (b) are HLA-DQA1 region genes.
The figure which shows the denaturation curve (melting curve) obtained about the known 0102 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

4 (a) and (b) are HLA-DQA1 region genes.
The figure which showed the denaturation curve (melting curve) obtained about the known 0103 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

5 (a) and (b) are HLA-DQA1 region genes.
The figure which showed the denaturation curve (melting curve) obtained about the known 0301 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

6 (a) and (b) are HLA-DQA1 region genes.
The figure which showed the denaturation curve (melting curve) obtained about the known 0401 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

7 (a) and (b) are HLA-DQA1 region genes.
The figure which showed the denaturation curve (melting curve) obtained about the known 0601 type single-stranded DNA from (242 bases or 239 bases) as the absorbance data and the differential absorbance data obtained by differentiating it with temperature.

FIG. 8 is a diagram in which a differential function of a 0301 type melting curve is approximated by superposition of Gaussian distribution functions.

FIG. 9 is a diagram showing a differential function of a denaturation curve (melting curve) and characteristic parameters of the curves in the case of heterozygous samples of 0102 type and 0301 type.

FIG. 10 is a diagram showing another analysis example of heterozygous DNA.

FIG. 11 is a diagram showing a measurement example when a denaturing curve (Melting Curve) draws a hysteresis curve.

FIG. 12 is a diagram showing a schematic flow of a process of an example of the present invention.

FIG. 13 is a diagram showing an outline of a device configuration of a first embodiment of the present invention.

FIG. 14 is a diagram showing a detailed configuration of a spectroscopic cell according to an example of the present invention.

FIG. 15 is a block diagram showing an embodiment of the configuration of a detection device for detecting fluorescence of DNA and intercalator.

FIG. 16: HLA-DQA1 * 0101 and DQA1 * 0
The figure which shows the result of the denaturation curve (Melting Curve) by the fluorescence of 102.

[Explanation of symbols]

1 ... Light source, 2 ... Optical system, 3 ... Ultraviolet light, 4 ... Sample, 5 ... Control, 6 ... Sample chamber, 7 ... Photoelectric converter, 8 ... Temperature controller, 9
... amplifier, 10 ... signal processing device, 201 ... cell, 202
… Sheath thermocouple, 300… Spectroscopic microcell, 301
... Temperature sensor (sheath thermocouple), 302 ... Temperature controller,
303 ... Sample outflow port, 600 ... PCR reaction cell, 60
1 ... Porous filter, 602 ... Primer A (solid phase), 7
01 ... PCR reaction solution supply valve, 702 ... Washing / spectroscopic buffer solution supply valve, 703 ... Waste liquid tank, 70
4 ... Waste liquid tank, 705 ... Door, 706 ... Door, 801, Light source, 802 ... Optical system, 803 ... Ultraviolet light,
804 ... sample, 805 ... sample chamber, 806 ... temperature controller,
Reference numeral 807 ... Optical system, 808 ... Photoelectric converter, 809 ... Amplifier, 810 ... Signal processing device.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References J. Mol. Biol (1994) Vol. 237, No. 5, p. 560-576 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12Q 1/68 C12M 1/00 C12N 15/00-15/90 BIOSIS / WPI (DIALOG)

Claims (20)

(57) [Claims] 1. A (1) preparing a single-stranded DNA fragment sample from the sample double-stranded DNA fragment, denaturing the (2) given denaturing conditions conformation of the sample single-stranded DNA fragments And (3) measuring denaturation curve data showing the relationship between the denaturation conditions and the result of denaturation of the higher-order structure
And (3) one having a known sequence and the above-mentioned denaturation curve data
Ratio comparing known denaturation curve data for single stranded DNA fragments
And comparing the sample single-stranded DNA with the comparison step.
Obtaining the sequence information of the fragment, the sample single-stranded DNA fragment
A method for gene analysis, which comprises analyzing a DNA polymorphism containing a single base substitution in Wherein (1) a step of preparing a single-stranded DNA fragment sample from the sample double-stranded DNA fragment, the base pairs that form a (2) the sample single-stranded DNA fragments conformation excitation irradiating the steps of intercalating a intercalator receives light emits fluorescent, the excitation Okoshiko single-stranded DNA fragment intercalated with (2) the intercalator
And (3) the sample single-stranded DN under irradiation of the excitation light
Denature the higher-order structure of the A fragment under predetermined denaturing conditions
Step, and (4) Before occurrence due to denaturation of the higher-order structure
The step of detecting a change in fluorescence intensity ; (5) measuring denaturation curve data showing the relationship between the denaturation conditions and the result of denaturation of the higher-order structure ; (6) the denaturation curve data ; Known degeneration of single-stranded DNA fragments with known sequence
It has a comparison process to compare with line data,
Sequence information of the single-stranded DNA fragment of the specimen from
Sample D containing single base substitution in single-stranded DNA fragment
A gene analysis method characterized by analyzing NA polymorphism . 3. The gene analysis according to claim 1 or 2.
In the method, the comparison step includes (a) the measured
Denaturation curve data and a plurality of known degeneration curve data
A single curve or a plurality of known denaturation curve data
Statistical error by comparing with curve prepared by linear combination
And (b) the statistical error is the smallest.
From the one curve or the curve resulting from the linear combination,
And a step of determining column information
Child analysis method. 4. The gene analysis according to claim 1 or 2.
In the method, the comparison step includes (a) the measured
Denaturation curve data and a plurality of known degeneration curve data
A single curve or a plurality of known denaturation curve data
Statistical error between pre-prepared linear combination curves
The difference is calculated, and the one song having the smallest statistical error
Selecting a line or a curve with said linear combination,
(B) The statistical error is less than a predetermined value.
The curve with the smallest
And determining the sequence information from the line.
Gene analysis method to determine. 5. The gene analysis according to claim 1 or 2.
In the method, the conformational denaturation is temperature denaturation.
Yes, the denaturation curve data is one sample for temperature
Obtained by change in absorbance or fluorescence intensity due to strand DNA fragment
A method for gene analysis characterized in that 6. The gene analysis according to claim 1 or 2.
In the method, the denaturing conditions are such that the higher order structure is formed.
Direction and the direction in which the higher-order structure can be solved alternately
Hysteresis of changes in absorbance or fluorescence intensity caused by changes
The sequence information was determined by measuring and analyzing cis.
A method for gene analysis characterized by: 7. The method for gene analysis according to claim 2.
The intercalator is ethidium bromide.
And a gene analysis method characterized by: 8. The gene analysis according to claim 1 or 2.
In the method, the comparison step includes (a) the measured
In advance by combining the denaturation curve data and a predetermined function
Statistical error compared to the known known denaturation curve data
And (b) the statistical error is the smallest.
Determining the sequence information from the known denaturation curve data
A method for gene analysis, comprising: 9. A pair of genes for selectively amplifying a specific gene region for analysis.
Reaction cell for producing single-stranded DNA fragment as elephant
And a spectroscopic cell holding a solution containing the single-stranded DNA fragment.
And transfer the solution from the reaction cell to the spectroscopic cell.
And the single-stranded DNA fragment in the solution
Spectroscopic means for measuring the absorbance of ultraviolet light, and the single-stranded DN
Denature the higher-order structure formed by the A fragment under prescribed denaturing conditions
Denaturing means and the denaturing conditions are set, and the denaturing means
The signal from the spectroscopic means is accumulated and the denaturing conditions and the
The denaturation curve data showing the relation with the result of denaturation of higher order structure
Single strand D having the above-mentioned denaturation curve data and known sequence
Comparing known denaturation curve data for NA fragments,
The sequence information of the DNA fragment is obtained, and
To analyze DNA polymorphisms containing single base substitutions in a piece
Signal processing means, and from the amplification as a pretreatment to the
The flow system is used continuously until the measurement of the denaturation curve data.
A gene analysis device characterized by performing. 10. The gene analysis device according to claim 9.
Multiple reaction cells into a single spectroscopic cell.
Connected to each of the reaction cells and generated in each of the reaction cells.
The solution containing the single-stranded DNA fragment is transferred to the spectroscopic cell.
A gene analyzer characterized by being introduced next. 11. A single-stranded DNA for a target gene region
The sample chamber that holds the sample containing it, and the purple that irradiates the sample chamber.
A light source that emits external light and the temperature of the sample chamber are programmed.
Control that changes along the specified temperature profile
Temperature controller and temperature sensor for measuring the temperature of the sample chamber
And a detector for detecting the ultraviolet light passing through the sample chamber
And the temperature of the sample chamber measured by the temperature sensor
And a signal processing device to which the signal from the detector is input.
The signal processing device has a higher-order structure of the single-stranded DNA.
The denaturation curve that represents the change in structure is
Corresponding to known polymorphism and processing to obtain as a change in luminous intensity
Known denaturation curve prepared in advance and the obtained denaturation curve
A process for comparing with a line is performed, and the single-stranded DNA is deleted.
A gene analysis device characterized by identifying a gene polymorphism. 12. A single-stranded DNA of a target gene region
The sample chamber that holds the sample containing it, and the purple that irradiates the sample chamber.
A light source that emits external light and the temperature of the sample chamber are programmed.
Control that changes along the specified temperature profile
Temperature controller and temperature sensor for measuring the temperature of the sample chamber
And a detector for detecting the ultraviolet light passing through the sample chamber
And the temperature of the sample chamber measured by the temperature sensor
And a signal processing device to which the signal from the detector is input.
And the signal processing device raises the temperature profile.
It is measured as a profile of temperature and subsequent temperature drop.
As a change in absorbance, the higher-order structure of the single-stranded DNA
A process for obtaining the hysteresis curve of a denaturation curve representing changes
And known pre-defined degenerate songs corresponding to known polymorphisms
Line hysteresis curve and hist of the denaturation curve obtained above
The single strand is processed by comparing with a telesis curve.
Gene characterized by specifying genetic polymorphism of DNA
Analyzer. 13. A single-stranded DNA of a gene region of interest
Inkers intercalated into base pairs that form higher order structures
The sample chamber that holds the sample, including the
The temperature of the sample and the light source that emits ultraviolet light
Control to vary along the rammed temperature profile
The temperature controller to be used and the
A detector for detecting fluorescence emitted from the calator, and the detection
And a signal processing device to which a signal from the vessel is input,
The signal processing device displays the change in the fluorescence intensity with temperature.
For the process of obtaining the denaturation curve and the known polymorphic DNA
Known denaturation curve prepared in advance and the obtained denaturation
The single-stranded DNA of the single-stranded DNA
Gene analysis device characterized by identifying gene polymorphism
Place 14. (1) Single chain D of a gene region of interest
The denaturation curve that represents the change in the higher order structure of NA
The process of measuring the change in absorbance due to (2) known
Known denaturation curves prepared in advance corresponding to polymorphisms and the above-mentioned total
And a step of comparing the measured denaturation curve,
Inheritance characterized by specifying gene polymorphism of strand DNA
Child analysis method. 15. The method for gene analysis according to claim 14.
In the step (2), the measured degeneration curve is measured.
By approximating the differential function of the line with a plurality of Gaussian functions,
The peak position and the width of the Gaussian function are defined by
The peaks of several Gaussian functions approximating the derivative function of the degenerate curve
Gene analysis characterized by comparing the position and width
Method. 16. The method for gene analysis according to claim 14.
In the step (2), the known denaturation curve
Differential function or linear combination of the differential functions of said known degenerate curves
And comparing the differential function of the measured degeneration curve with
A gene analysis method characterized by: 17. (1) Single chain D of a target gene region
Changes in the higher-order structure of NA are caused by temperature increase and subsequent temperature decrease.
Hysteresis curve of denaturation curve obtained as change in absorbance
A step of measuring the line, and (2) corresponding to a known polymorphism in advance
The prepared hysteresis curve of the known denaturation curve and the above
Comparing the measured degeneration curve with the hysteresis curve
And identifying the gene polymorphism of the single-stranded DNA.
And a gene analysis method characterized by: 18. (1) Single chain D of a gene region of interest
Intercalated into base pairs that form the higher order structure of NA
Denaturation showing changes in fluorescence intensity of incalator with temperature
For the process of measuring the curve and (2) known polymorphic DNA
Known denaturation curve prepared in advance and the measured denaturation
Identifying the polymorphism of the single-stranded DNA by comparing with a curve
The method for gene analysis, comprising: 19. The gene analysis device according to claim 11.
And, the signal processing device is
The process of approximating the differential function with a plurality of Gaussian functions,
The peak position and width of the Gaussian function of
Of multiple Gaussian functions approximating the derivative function of a known degenerate curve
Characterized by the process of comparing the peak position and width
Gene analysis device. 20. The gene analysis device according to claim 11.
The signal processing device is a derivative of the known degeneration curve.
Function or linear combination of the derivative functions of said known degenerate curve and
It is special to compare with the derivative function of the obtained degeneration curve.
Gene analysis device to collect.