JPH0674936A - Nucleic acid or protein analysis method and analyzer - Google Patents

Abstract

(57) [Summary] [Objective] To perform high-speed separation of a small amount of DNA of several thousand bases or more by capillary gel electrophoresis using agarose gel and on-column detection. [Arrangement] An electrophoretic section 2 comprising a glass capillary filled with agarose gel for separating a sample by an electrophoretic method,
The high-voltage power supply unit 1 for applying a high voltage to the electrophoretic unit 2, the driving unit 3 for moving the tip of the glass capillary of the electrophoretic unit 2 to the sample container 8 to inject the sample, and the driving unit 3 x
Separated by a controller unit 4 for controlling the operation in the yz direction, a connecting unit 6 for connecting a glass capillary for an electrophoresis unit filled with agarose gel and a glass capillary for an optical detection unit filled with a polyacrylamide gel, and an electrophoresis unit 2. It is composed of an optical detection section 5 for detecting the detected component and a data processing section 7 for processing the data detected by the optical detection section 5.

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 nucleic acid or protein using capillary electrophoresis, or an analyzer.

[0002]

2. Description of the Related Art Capillary electrophoresis is superior to high performance liquid chromatography in that it can perform analysis with a small amount of sample of 1/1000 or less, and has several tens of times higher separation performance.

Further, as compared with the conventional slab electrophoresis or the like, it is possible to shorten the analysis time by being able to apply a high voltage. Further, since one end of the capillary can be used as an injector, the sample injection can be easily automated.

Therefore, in recent years, the capillary electrophoresis method has been widely used in many fields. In particular, it is being applied to nucleic acid and protein analysis with the development of bio-related research. A UV detection method or a fluorescence method is generally used as a method for detecting nucleic acids and proteins.

In the capillary electrophoresis method, when the UV detection method or the fluorescence method is used, the on-column detection method in which the capillary and the detector are integrated is widely used.

[0006] In analysis of nucleic acids, particularly in sequencing, capillary gel electrophoresis using polyacrylamide is usually used, and sequencing of about 300 bases is performed in about 45 minutes. Furthermore, although there is a report that a DNA fragment of about 10,000 bases was separated in 20 minutes, the separation ability was 1,000 base units at several thousands bases or more. For the capillary electrophoresis method, see Science,
Volume 222 (1983) 266 to 272 tributes (S
science, Vol. 222 (1983) pp. 26
6-272), for nucleotide sequencing, see Analytical Chemistry, Vol. 62 (1990) No. 900 to No. 903 (Analytical Chemist).
ry, Vol. 62 (1990) pp. 900-90
3), for the analysis of a DNA fragment of about 10,000 bases, see Journal of Chromatography 516.
Volume (1990) 33rd to 48th tributes (Journa
lof Chromatography, Vol. 51
6 (1990) pp. 33-48).

[0007]

In the on-column detection method in which a capillary and a detector are integrated, which has been used as a conventional method for detecting nucleic acid or protein, an optical path is provided in a part of the capillary for detection. Generally, in the slab electrophoresis method, an agarose gel is used for separating DNA of 1000 bases or more. Therefore, it was necessary to use agarose gel instead of polyacrylamide gel for the separation of DNA of several thousand bases or more.

However, the agarose gel has a poor light transmittance and is not suitable for the on-column detection method. Therefore, it has been very difficult to separate DNA of several thousand bases or more by capillary electrophoresis.

Further, the agarose gel has a problem in heat resistance. Particularly, in the capillary gel electrophoresis method, since a high voltage is applied, the Joule heat generated in the capillary becomes a big problem, and it is very difficult to use the agarose gel.

[0010]

In order to solve the above problems, in the present invention, when a DNA of several thousand bases or more is analyzed by capillary electrophoresis, agarose gel is used for separation. The on-line detection was performed with the detection unit filled with polyacrylamide gel having excellent light transmittance. At that time, as a more specific device, (1) The agarose gel and the polyacrylamide gel were connected using a glass connector, and the polyacrylamide gel was used to seal the connection part to pass the separated sample. , And conductivity was maintained.

(2) The value of the current flowing in the capillary is suppressed to suppress the heat generation of Joule heat generated in the capillary.

[0012]

By using an agarose gel for the capillary electrophoresis method, it is possible to easily separate DNA of several thousand bases or more. Since a polyacrylamide gel having excellent light transmittance is used for the detection unit, the DNA separated by the agarose gel can be optically detected by the on-column detection method while maintaining high resolution. In addition, it is possible to suppress the generation of Joule heat by appropriately selecting the buffer concentration.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the block diagram of FIG. The capillary gel electrophoresis apparatus according to the present invention connects the high-voltage power supply unit 1, the electrophoresis unit 2, the driving unit 3, the controller unit 4 for controlling the driving unit 3, the optical detection unit 5, the electrophoresis unit 2 and the optical detection unit 5. Connection part 6,
And a data processing unit 7 for processing the data detected by the optical detection unit 5. The high-voltage power supply unit 1 uses an output voltage of 0 to 30 kV and a high-voltage power supply whose polarity can be easily switched.
0 and 11, and electrodes 13 and 11 between the buffer tank 12 and the buffer tank 9 arranged in parallel in the sample container 8.
Is used to apply voltage. The drive unit 3 is driven by a pulse motor to control the movement in the xyz directions. The controller unit 4 controls the operation in the xyz directions by the drive unit 3, and general-purpose operations such as sample suction can be performed by programming a moving distance in the xyz direction and a stop time with one switch. it can.

To inject the sample into the capillary gel electrophoresis apparatus, first, the tip of the glass capillary of the electrophoresis section 2 is moved to the sample container 8 and the high voltage power supply section 1 causes the electrode 1 to move.
By applying a voltage with a predetermined polarity to 0 and 11, the sample in the sample container 8 was aspirated by a fixed amount of the sample from the tip of the glass capillary of the electrophoresis section 2 by electrophoresis. The amount of sample to be sucked can be easily determined by the applied voltage and the voltage application time even in the picoliter order.

After that, the tip of the glass capillary of the electrophoresis section 2 was moved to the buffer tank 12, and a predetermined voltage was applied to the electrodes 13 and 11 to perform electrophoresis.

In the present invention, the electrophoretic section 2 has an inner diameter of 50.
A fused silica capillary with a diameter of 50 μm, an outer diameter of 375 μm and a length of 50 cm, and a 0.3% agarose gel were used. The agarose gel concentration can be selected according to the molecular weight of the components to be separated.

The optical detection section 5 has 3% T (weight percentage of polyacrylamide monomer), 0.5% C (weight percentage of bispolyacrylamide in polyacrylamide).
A glass capillary filled with polyacrylamide gel was used.

For the preparation of the polyacrylamide gel, N, N, N ', N'-tetramethylethylenediamine and ammonium peroxodisulfate as polymerization agents were used.
In case of separating DNA of several thousand bases or more, 5
It is desirable to use less than% T polyacrylamide.

As the optical detector 5, an ultraviolet absorption detector for HPLC improved for on-column detection was used, and the absorbance at a wavelength of 260 nm which is the measurement wavelength of DNA was measured. The measured data from the optical detector 5 was sequentially transmitted to the data processing unit 7, and the data processing unit 7 recorded and processed the data.

FIG. 2 shows an example of analysis of the DNA fragments obtained by the example of the present invention. Table 1 shows the measurement conditions. The DNA fragments used here are T4dC * -DNA and T4dC-D which are commercially available as DNA molecular weight markers.
It can be seen that the mixture of NA was completely digested with the restriction enzyme Bgl I, and this could be completely separated within 30 minutes.

[0021]

[Table 1]

Next, the case of connecting the glass capillary filled with agarose gel and the optical detection unit filled with polyacrylamide gel, which is required in the embodiment of the present invention, will be described with reference to FIG.

A glass capillary 14 for an electrophoresis section filled with agarose gel and a glass capillary 15 for an optical detection section filled with polyacrylamide gel were individually prepared. Then, the glass capillaries 14 and 15 each filled with gel are inserted and fixed in the glass capillary connector 17 fixed in the joint 16 for connection.
Fixing is performed by pressing tools 18, 19 and push screws 20, 21.
Was used to apply force to one end of each glass capillary on the same straight line. It is even more preferable to fill the inside of the glass capillary connector 17 with polyacrylamide gel in advance and insert and fix both capillaries in such a manner as to push it out.

Another embodiment in which a glass capillary for an electrophoresis section filled with agarose gel and a glass capillary for an optical detection section filled with polyacrylamide gel are connected will be described with reference to FIG.

Grooves 24 and 25 for glass capillaries are provided on the quartz glass plates 22 and 23. The method of forming the groove is
Machining or chemical etching can be used. A glass capillary 26 for an electrophoresis section filled with agarose gel and a glass capillary 27 for an optical detection section filled with polyacrylamide gel were individually prepared. After that, the glass capillary 26 for the electrophoretic section and the glass capillary 27 for the optical detection section were mounted in the groove 25 on the quartz glass plate 23, and the quartz glass plate 22 was placed in close contact therewith. A force for pushing the quartz glass plates 22 and 23 vertically is applied by a spring 28 and an outer box 29. It should be noted that the two quartz glass plates are in close contact with each other by a polytetrafluoroethylene seal, and there is no leakage between the glass capillaries. Further, a polytetrafluoroethylene plate can be used instead of the quartz glass plate. Also in this case, it is preferable to apply polyacrylamide gel between the quartz glass plates and between the polytetrafluoroethylene plates in advance, and connect them in such a manner as to eliminate them.

Next, the calorific value and the buffer will be described.

In general, the current flowing in a solution depends on the ions present in the solution. Therefore, the inventor of the present application considered that the current value flowing through the capillary was influenced by the buffer used, and investigated the relationship between the applied voltage and the heat generation amount in various buffers. The result is shown in FIG. The buffers used for the measurement each have a concentration of 80 mM and a pH of 8.3. In addition, the measurement is the inner diameter
0.3% in a 5 μm, 50 cm long glass capillary
It was performed using the one filled with agarose gel.

As a result, the amount of heat generation is Tris-
phosphate>Tris-HCl> Tris-a
It becomes smaller in the order of cerate> Tris-borate, and Tris-bora with the smallest calorific value among them becomes smaller.
The amount of heat generated when te buffer was used was Tris-p.
It was about 1/5 of the case of phosphate.

Further, when a glass capillary having an inner diameter of 50 μm and a length of 50 cm is used, the calorific value is 75 μm.
Approximately 1 / m in case of glass capillary with m and length of 50 cm
It was 3. Further, from the series of experimental results shown in FIGS. 2 and 5, it was found that measurement was possible when the calorific value was 0.06 W / m or less.

From the above, in order to suppress the current value flowing through the capillary, it was judged that Tris borate buffer was advantageous as a buffer, and the relationship between the applied voltage and the calorific value at various borate buffer concentrations was investigated, and the results are shown in FIG. 5 shows. The measurement is 50 μm in inner diameter and 50 in length.
cm glass capillaries filled with 0.3% agarose gel were used.

As a result, the calorific value was proportional to the buffer concentration, and when the buffer concentration was halved, the calorific value was halved. Therefore, the calorific value generated in the capillary is reduced to 0.
It was found that, in order to suppress the concentration to 06 W / m or less, the concentration of the tris borate buffer should be 80 mM or less in the case of an applied voltage of about 150 V / cm which is generally used. Since the buffer also has a function of maintaining the pH of the sample, it is not necessarily enough if it is thin, and according to the experiments of the inventor, it was necessary to be 10 mM or more.

When the applied voltage is 200 V / cm or more, there is no problem if the trisborate buffer concentration is 40 mM or less.

Further, by using a glass capillary having an inner diameter of 50 μm or less, the ratio of the outer surface area where heat is radiated to the volume of the glass capillary where Joule heat is generated is increased, and the heat radiation efficiency is improved.

[0034]

According to the examples of the present invention, since the agarose gel is used for the electrophoretic section and the polyacrylamide gel excellent in light transmission is used for the detection section, DNA of several thousand bases or more is used. The on-column detection method can be used to easily analyze macromolecules.

[Brief description of drawings]

FIG. 1 is a block diagram showing the configuration of a nucleic acid or protein analyzer according to an embodiment of the present invention.

FIG. 2 is a diagram showing analysis of DNA fragments obtained according to the examples of the present invention.

FIG. 3 is a cross-sectional view showing a connection configuration of an electrophoretic unit and an optical detection unit that can be used in an embodiment of the present invention.

FIG. 4 is a cross-sectional view showing a connection configuration of an electrophoretic section and an optical detection section provided on a quartz glass plate.

FIG. 5 is a diagram showing the results of experiments on the relationship between the amount of heat generated by various buffers and applied voltage.

FIG. 6 is a diagram showing the results of experiments on the relationship between the concentration of borate buffer and the amount of heat generated by applied voltage.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... High-voltage power supply part, 2 ... Electrophoresis part, 3 ... Driving part, 4 ... Controller part, 5 ... Optical detection part, 6 ... Connection part, 7 ... Data processing part, 8 ... Sample container, 9, 12 ... Buffer tank 1
0, 11, 13 ... Electrodes.

Claims (8)

[Claims] 1. A nucleic acid or protein characterized by optically detecting at a portion of a polyacrylamide gel housed in a capillary after separation by electrophoresis on an agarose gel housed in a capillary. Analysis method. 2. An agarose gel housed in a capillary and a polyacrylamide gel housed in the capillary are composed of an electrophoresis section arranged in a cascade, and an optical detection section provided in the polyacrylamide gel section. Nucleic acid or protein analyzer. 3. An apparatus for analyzing nucleic acids or proteins, characterized in that, in a capillary electrophoresis apparatus having an agarose gel electrophoresis section, a borate buffer is used as a buffer to be supplied to the capillary after the sample is supplied to the agarose gel. 4. The method or analysis of nucleic acid or protein according to claim 1, wherein the capillary is performed using a glass capillary having an inner diameter of 100 μm or less. apparatus. 5. The connection is made by arranging one end of the glass capillary filled with the agarose gel and one end of the optical detection portion filled with the polyacrylamide gel on the same straight line. Alternatively, the method for analyzing nucleic acid or protein according to claim 2, or the analyzer. 6. The method for analyzing a nucleic acid or protein according to claim 1 or 2, or an analyzer, wherein the agarose gel electrophoresis and the optical detection are performed on the same plane. . 7. A glass connector is used to connect the glass capillaries filled with agarose gel and polyacrylamide gel, respectively, and the polyacrylamide gel is put on the outer periphery of the connecting portion of the glass capillary and the inner surface of the glass connector. The nucleic acid or protein analyzer according to claim 2, which is characterized in that. 8. The nucleic acid or protein analyzer according to claim 3, wherein the borate buffer concentration is 80 mM or less and 10 mM or more.