JPH06138090A - Method and instrument for analyzing multiple genotypes using capillary electrophoretic method - Google Patents

Abstract

PURPOSE:To analyze multiple genotypes with high resolution in a short time by using a gel housed in a capillary having an inside diameter smaller than a prescribed value for electrophoretic analysis. CONSTITUTION:When a capillary electrophoretic method for on-column detection is used for an electrophoretic and detecting means, the electrophoretic pattern of a sample which should indicate multiple higher-order structures can be detected as sufficiently separated two peaks. Regarding the amount of a single- stranded DNA per one band, accurate pattern analysis can be performed, because the amount of the band separately detected from bands detected in an overlapping state due to the higher-order structures can be directly read as numerical values from the heights of peaks. In addition, the temperature controlling accuracy of an electrophoretic carrier is also improved. When, for example, the volume (V/S) corresponding to the calorific value generated per unit heat radiating area is taken into consideration, a heat radiating effect which is about 20 times as high as that of a slab gel having a 1mm thickness (V/S=0.5) is obtained when a capillary of 100mum (V/S=0.025mm) in diameter is used. The required time can be also reduced to 1/6 to 1/10.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing genetic polymorphisms which is useful for genetic diagnosis or genetic testing necessary for medical treatment.

[0002]

2. Description of the Related Art Polymorphisms accumulated in genes of humans and other organisms contain a lot of medical information in association with genetic diseases and immune mechanisms, and a rapid and accurate analysis method thereof is desired. .

[0003] For example, more than 400 polymorphisms are accumulated in the histocompatibility complex (HLA) gene, which is very closely related to the immune system, and 100 polymorphisms are accumulated by combining them.
There are over 100 serotypes. This serotype determination is a particularly important problem such as in the case of organ transplantation, and it has been suggested that it is associated with many diseases. Therefore, HLA serotype determination will become very important in the field of diagnosis and medical examinations in the future. Is coming.

Further, the detection of single nucleotide level polymorphism
It means the detection of mutations in gene sequences and leads to the diagnosis of many diseases including cancer. It has been suggested that gene mutations contribute to the mechanism of carcinogenesis and metastasis, and if it is possible to quickly determine the classification of gene polymorphisms and mutations in a large number of samples, it will be very useful for diagnosis and identification of cancer types. Conceivable.

As a means for detecting the gene polymorphism at the single nucleotide level with high sensitivity, a method for analyzing the nucleotide sequence polymorphism of DNA by utilizing the difference in the higher-order structure of single-stranded DNA (Single Strand Conformation Polymorphism; hereinafter abbreviated as S.C.P. method) is the Proceeding of National Academy of Science of USA.
regionalAcademy of Science
of U. S. A. ) Vol. 86, pp2766-2
770 (1989). Proposed by Orita et al.

In this method, a target portion on a DNA base sequence extracted by an appropriate method is dissociated into a set of single-stranded DNAs complementary to each other by an appropriate denaturing means, and they are non-denaturing polyacrylamide gel. Electrophores in.
At this time, the migration rate of each single-stranded DNA changes under the influence of the higher-order structure of the single-stranded DNA, and this higher-order structure is specifically determined by the sequence of the single-stranded DNA. By utilizing this property, a sequence polymorphism due to a difference of at least one base is detected.

[0007] This method is very simple, and moreover, it is possible to detect polymorphisms due to the difference of one base with high sensitivity. Therefore, the restriction enzyme cleavage polymorphism analysis method previously invented,
It is considered to be very advantageous as compared with the polymorphism analysis method using a sequence-specific DNA probe, and recently, in combination with the nucleic acid amplification method (hereinafter abbreviated as PCR method), it is often used for polymorphism analysis and identification of gene mutation. It is used.

[0008]

However, the conventional S.M. S.
C. In the P. method, since electrophoresis was performed using a slab gel, it was difficult to achieve high resolution / quantification of the electrophoretic pattern, which is important information for polymorphism analysis. In addition, migration and detection take time, reassociation of double strands occurs while loading the gel after denaturing the sample into single strands, which has an important effect on the maintenance of higher-order structure. Difficult to control,
There were also problems such as.

Therefore, the object of the present invention is to S. C. P.
It is an object of the present invention to provide a method for performing gene polymorphism analysis by the method more easily than ever before, with high resolution and in a short time.
Also, S. S. C. It is also an object of the present invention to enable automation of polymorphism analysis by the P. method.

[0010]

In order to achieve the above object, in the present invention, the capillary electrophoresis method of on-column detection is used as the electrophoresis / detection means. Further, a constant temperature bath capable of heating the sample to the dissociation temperature at the time of injection was attached to the sample stage so that the injection could be directly performed in the gel at the dissociation temperature. Furthermore, the capillary temperature during the migration was controlled by a thermostat plate.

[0011]

[Function] By using the capillary electrophoresis method of on-column detection as the electrophoresis / detection means, the electrophoresis pattern of the sample that should show higher-order structure polymorphism can be detected as two sufficiently separated peaks. It was Regarding the amount of single-stranded DNA per band, the quantification of the band detected by separating from the band detected by overlapping with the higher-order structure is
It became possible to read directly as a numerical value by the height of the peak, and it became possible to perform pattern analysis accurately.

Using capillary electrophoresis, S. S.
C. Performing P. analysis makes it possible to easily improve the accuracy of temperature control of the electrophoretic carrier. Volume / area ratio (V / S ratio) corresponding to the amount of heat generated per unit heat dissipation area
In comparison, the slab gel with a thickness of 1 mm (V / S =
When a capillary having a diameter of 100 μm (V / S = 0.025 mm) is used, the heat radiation efficiency is about 20 times higher than that in the case of performing electrophoresis using 0.5 mm).

S. S. C. The polymorphism separation principle of the P. method is to carry out polymorphism separation by utilizing the fact that the stable higher-order structure in a given temperature condition is different due to the difference in arrangement. That is, S. S. C. In the P. method, since it is necessary to maintain a temperature-dependent sequence-specific higher-order structure during electrophoresis, it is a very important condition that the electrophoresis carrier has a uniform temperature during electrophoresis. From this point as well, S. S. C. If P. is carried out, it becomes possible to separate and detect sequence polymorphisms that could not be separated in the past.

The advantage of using a Peltier element in the temperature control plate of the capillary is also great. By changing the polarity of the applied voltage, the Peltier element can realize both cooling and heating with one element. In an apparatus such as the present invention which has a heat source called a capillary gel and requires accurate temperature control to a temperature near room temperature, cooling and heating are selectively controlled at arbitrary times as described above. Being able to do is an important point. This enables temperature control with an accuracy of the set temperature ± 0.1 ° C over the entire range of 5 ° C to 60 ° C.

The time required for electrophoresis is the same as that of S. S. C. Compared with 2 hours to 5 hours or more in the case of P. method, it was greatly shortened to 1/6 to 1/10.

[0016] Capillary electrophoresis is capable of sufficiently migrating and detecting even a minute amount of sample. In the case of capillary electrophoresis with absorbance detection by ultraviolet light, the concentration is 5 x 1
/ 10 ⁸ mol / L, 5 × 1/10 ¹⁹ grams in absolute amount can be sufficiently detected. It can be said that these amounts are sufficiently good sensitivities even compared with the detection sensitivities of conventional slab gels. If higher sensitivity is required, it is possible to increase the sensitivity with a fluorescent label.

Further, by heating the sample to the DNA dissociation temperature and introducing it into the gel while the DNA is dissociated into single strands, all sample DNAs are electrophoresed as single strands.

In the case of electrophoresis using a conventional slab gel, since the sample introduction part to the gel which is the electrophoretic carrier is always in the buffer tank, it is quite difficult to heat it above the DNA dissociation temperature, and it is almost the same as the gel. Since the sample is introduced into the gel via the migration buffer at the temperature, there was a problem that a reassociation reaction between complementary strands occurs at the time of introduction into the gel, or an appropriate denaturant is required at the time of introduction of the gel. . This is because the temperature at which a stable higher-order structure is formed in the molecule is in a temperature region where reassociation is likely to occur sufficiently.

However, in the case of the capillary electrophoresis method, since the sample can be preheated to the DNA dissociation temperature before the introduction, the above problems can be solved. This is because in the capillary electrophoresis apparatus used in the present invention, the sample container and the migration buffer are separate and the tip of the capillary is brought into contact with the sample solution to introduce the sample only when the sample is introduced. This is because it is possible to heat up to.

As a result, the band of the double-stranded DNA due to the halfway reassociation reaction does not disturb the detection peak, so that not only a sharp migration pattern can be obtained but also all the introduced DNAs can be obtained. Since it is possible to detect the higher-order structure polymorphism of, the polymorphism can be sufficiently detected even by introducing a small amount of sample DNA.

[0021]

EXAMPLE An example of the present invention will be described below by using an example of detecting a mutation sequence at the single base level.

The target polymorphism site to be detected is human fa
A 171 bp region was selected from the ctorΙΧ gene.
It is known that there are 12 types of polymorphisms due to point mutations at this gene site (Gobinda Sar
kar et. al; Nucleic Acids
Research, Vol. 20, No. 4, p
871-878).

The analyzed human DNA is Maniatis
Method (Molecular Cloning p 28
0-281 (1982)). 1 μg of the above genomic DNA was added to 100 μl of 50 mM
KCl, 10 mM Tris-HCl (pH 8.3),
200 μM dA each in 1.5 mM MgCl 2 solution
TP, dCTP, dGTP, dTTP, 20 pmol:
It was dissolved with an oligonucleotide primer prepared at a molar ratio of 20 pmol, in which a polymerase (Taq-Polymeras from Thermus aquaticus) was dissolved.
2.5 unit of e) was added. After overlaying this with 40 μl of mineral oil, 2 minutes at 92 ° C, 2 minutes at 55 ° C, 2 minutes at 72 ° C.
The double-stranded DNA at the target site was specifically amplified by repeating the 40 minute cycle. After removing the mineral oil from this reaction solution, 250 μl of 5M CH ₃ COONH
₄ Add ethanol and centrifuge at 0 ° C., 14,000 × g for 30 minutes, remove the upper layer solution (ethanol precipitation), wash the precipitate (pellet form) with 80% ethanol (ethanol rinse; desalting), 5 times the dried precipitate
0 μl of 10 mM Tris-HCl (pH 7.9),
It was dissolved in a 1.0 mM EDTA, 20 mM NaCl solution, and the solution was subjected to capillary electrophoresis.

FIG. 1 is a block diagram showing the capillary electrophoresis apparatus used in this embodiment. The capillary electrophoresis apparatus according to the present invention includes a high voltage power supply section 1-1, a capillary 1-2, a sample holding container / sample temperature adjusting plate 1-3,
Capillary holding part and capillary temperature control plate 1
-4, a drive unit 1-5, buffer tanks 1-6 and 1-7, an optical detection unit 1-8, and a data processing unit 1-9 that processes data detected by the optical detection unit 1-8. The sample temperature control plate 1-3 and the capillary temperature control plate 1-4 are independent temperature controllers 1-10 and 1-11, respectively.
, Especially capillary temperature control plate 1-1
1 is 5 ℃ to 6 with an accuracy of ± 0.1 ℃ in the direction of the capillary axis.
It is configured to be adjustable in the range of 0 ° C.

Further, the capillary temperature control plate in the present invention controls the temperature of the aluminum plate 1-12 by the Peltier element 1-13, and either the cooling or the heating is performed by changing the polarity of the voltage applied to the Peltier. Will also be possible. Sample temperature control unit 1-3 and 1-1
0 is configured so that the sample can be heated up to the DNA dissociation temperature. Although not performed in this embodiment, it is possible to use a Peltier element or the like so that the sample can be introduced at an ice cooling temperature.

The high voltage power supply section 1-1 has an output voltage of 0 to 30.
Using a high voltage power supply with kV and easy polarity switching,
Electrode 1 between sample container 1-3 and buffer tank 1-7
14, 1-15, electrode 1-1 between the buffer tank 1-6 and the buffer tank 1-7, which are attached to the sample container 1-3
6, 1-15 are used to apply the voltage.

The sample is introduced into the capillary gel electrophoresis apparatus by first heating the sample to a predetermined temperature and then attaching the tip of the glass capillary (1-2) of the electrophoresis section to the sample container 1-.
Then, the sample in the sample container was electrophoretically introduced into the glass capillary 1-2 by applying a voltage of a predetermined polarity to the electrodes 1-14 and 1-15. In this case, by controlling the applied voltage and voltage application time,
It is possible to control the amount of sample introduced in the picoliter order. One picoliter is approximately one millimeter in length of the capillary.

Then, the tip of the glass capillary of the electrophoresis section is moved to the buffer tank 1-6, and the electrodes 1-16, 1
Electrophoresis was performed by applying a predetermined voltage between −15.

The operating conditions used in this example are summarized in the table below.

[0030]

[Table 1]

However, the working conditions used in this embodiment are merely examples, and the gel composition, the buffer composition, the applied voltage, the set temperature, etc. are not particularly limited to the present invention.

Next, the results of electrophoresis detection of this example are shown in FIGS.

FIG. 2 shows the results of electrophoresis when a sample having a mutation was subjected to direct electrophoresis after amplification of the target region by PCR without denaturation and dissociation process into single strands. FIGS. 3 and 4 show the target region. After being amplified, it was dissociated into single strands and subjected to electrophoresis. The vertical axis on the data is the absorbance (abs), and the horizontal axis is the migration time (minute) after the introduction. here,
2 and 3 are the results of migration of normal samples, and the samples shown in FIG. 4 are the results of migration of heterozygous samples of normal DNA and mutant DNA.

In FIG. 2, only one peak is detected at the position of 16 minutes, which is a peak of double-stranded DNA of 171 bp in comparison with the migration results of other molecular weight markers.

On the other hand, in FIG. 4, which is a result of electrophoresis after dissociation into single strands, four peaks were detected in 16 to 19 minutes. Regarding two of these peaks,
The same peak of normal DNA as that detected in FIG. 3 is also obtained. The other two peaks are due to the DNA having a mutation.

As described above, according to the present invention, it becomes possible to detect the presence or absence of a mutation of at least one base on a gene very easily and clearly in a short time.

Although not carried out in this embodiment, by using the capillary electrophoresis apparatus as described above, the S. S. C. It is also possible to perform P. analysis. S. S. C. Separation of polymorphisms in the P. method is sensitive to temperature conditions, and when a new polymorphism is separated, it may take considerable time to determine optimal temperature conditions. In slab gel, it is difficult to perform electrophoresis at various temperatures at the same time because of problems such as enlargement of the equipment, but in the case of capillary electrophoresis equipment, it is necessary to perform electrophoresis under different conditions at the same time and find the optimum conditions in a short time. Will be possible.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, a gene polymorphism due to a difference in sequence of at least one base or more is used for the S. S. C. Compared with the P. method, the detection can be performed more accurately and more easily in a considerably shorter time. In addition, although it is useful as a high-throughput means for gene mutation diagnosis and polymorphism analysis, it has been difficult to automate S. S. C. By implementing the P. method as in the present invention, it is considered that the possibility of automation greatly expands.

As a result, it is possible to contribute to the development of the field of gene diagnosis for cancer and the field of genetic polymorphism test performed at the time of organ transplantation.

[Brief description of drawings]

FIG. 1 is a block diagram of a capillary electrophoresis apparatus used in this example.

FIG. 2 is a diagram showing an example of a migration result of a normal sample (double-stranded) in this example.

FIG. 3 is a diagram showing an example of the S.C.P. analysis result of a normal sample in this example.

FIG. 4 shows the S.S. of specimens having a mutation in this example.
Figure showing an example of CP analysis results

[Explanation of symbols]

1-1 ... High-voltage power supply, 1-2 ... Glass capillary, 1-3 ... Sample container and sample temperature control plate, 1-4 ... Capillary temperature control plate, 1-5 ... Capillary drive device, 1-6
… Buffer tank, 1-7… Buffer tank, 1-8… Optical detector, 1-9… Data processing device, 1-10… Sample container temperature controller, 1-11… Capillary temperature controller, 1-12… Aluminum plate, 1-13 ... Peltier element, 1-14 ... electrode, 1-15 ... electrode, 1-16 ... electrode.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location 7235-2J G01N 27/26 315 Z

Claims (4)

[Claims] 1. A double-stranded DN containing a biological or viral nucleic acid.
For a predetermined region of A, the DNA region is dissociated into single-stranded DNAs complementary to each other, and the DNA is electrophoresed by gel electrophoresis,
A method for analyzing a sequence polymorphism of a DNA by utilizing a higher-order structure polymorphism that single-stranded DNA specifically takes in the sequence polymorphism,
A method for analyzing nucleic acids, characterized in that the electrophoresis analysis is performed by an electrophoresis method using a gel housed in a capillary having an inner diameter of 100 μm or less. 2. The method for analyzing a nucleic acid according to claim 1, wherein electrophoresis and detection are performed while controlling the capillary at a constant temperature. 3. The method for analyzing nucleic acid according to claim 1, wherein the DNA sample is directly loaded into the capillary gel in a state of being heated to a temperature higher than the dissociation temperature of DNA. 4. A temperature adjusting device for accommodating a sample and heating the sample to a predetermined temperature, a capillary for performing electrophoresis, a temperature controller for controlling the capillary to a predetermined temperature, and a high temperature device at both ends of the capillary. A power supply device that can apply a voltage, a drive device that moves the capillary tip within a predetermined range, an optical detector that detects a sample that migrates in the capillary, and a signal that is output from the detector. And a processing device for the nucleic acid analysis device.