JPH0750073B2 - Capillary electrophoresis - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for separating and analyzing nucleic acid and the like, and particularly to a capillary electrophoresis method suitable for separating components having similar properties of such substances.

[Prior Art] Conventionally, agarose electrophoresis, polyacrylamide gel electrophoresis and the like have been widely used for separation and analysis of nucleic acids and the like. However, in these methods, the gel is basically disposable, and it takes time to prepare the gel, which is disadvantageous especially in automating the system.

In recent years, attempts have been made to apply capillary electrophoresis to separation analysis of nucleic acids or proteins. In particular, the application to nucleic acids has been reported by BL Karger et al. Or RGBrownlee et al.

Capillary electrophoresis is performed without a carrier,
Karger et al. Separated the mixture of DNA restriction fragments using fused silica as a capillary and a tris (hydroxymethyl) aminomethane-borate buffer containing 7M urea and 0.1% sodium dodecyl sulfate as a running buffer. (Journal of Chromatography, 458 (198
8) 323-333). Brownlee et al. Used NaH ₂ containing 4M urea and 20 mM cetyltrimethylammonium bromide as a running buffer using fused silica as the capillary.
PO ₄ -Na ₂ B ₄ O ₇ with buffer separating the mixture of DNA restriction fragments (Journal of Chromatography, 458
(1988) 303-312). However, in both cases, the mechanism of DNA separation is not clear, and in the former case, a peak of unknown origin may appear, and it is difficult to reproduce the separation due to a slight difference in sample pretreatment and injection conditions. There is a drawback that In the latter case, sufficient separation has not been obtained.

For capillary electrophoresis performed by filling the gel with the carrier as a carrier, Brownlee et al. Used a capillary filled with a 3% T, 5% C polyacrylamide gel as a DN.
Isolating a mixture of A restriction fragments (Journal of Ch
romatography, 458 (1988) 303-312). Karger et al separates the mixture of d (A) ₄ θ _-6 θ oligonucleotides as well (Pro.Natl.Acad.Sci.USA, 85 (1988) 9660
-9663). However, in capillary electrophoresis using a polyacrylamide gel, since the inner diameter of the capillary is small, a polyacrylamide gel cannot be formed in the capillary with good reproducibility, and the reproducibility of separation of the mixture is poor. Another method using a gel as a carrier is to use an agarose gel (Brownlee et al., Journal.
al of Chromatography, 458 (1988) 303-312), agarose gel is physically weak and flows out during electrophoresis.
In addition, the gel is melted and flows out from the capillary due to a temperature rise caused by heat generation during electrophoresis. For these reasons, when electrophoresis is performed using a capillary filled with agarose gel, the mixture cannot be separated with good reproducibility.

From this, the present inventor has added a non-gelled agarose-based polymer to the capillary electrophoresis buffer,
We invented a capillary electrophoresis method that separates with good reproducibility (patent pending), and have performed separation analysis of DNA and the like. However, the size range of DNA that can be separated by this method is 10 to 1
As narrow as 000 base pairs, it was difficult to separate particularly large DNA.

[Problems to be Solved by the Invention] When attempting to perform separation and analysis of nucleic acids and the like, agarose electrophoresis and polyacrylamide gel electrophoresis pose problems of gel preparation labor and automation, and When separation and analysis is performed by capillary electrophoresis, it is a problem that DNA in a wide range of sizes cannot be sufficiently separated even in a system using an agarose polymer that is not gelled.

[Object of the Invention] An object of the present invention is to perform capillary electrophoresis in place of agarose electrophoresis or polyacrylamide gel electrophoresis, using a buffer for electrophoresis in which the concentration of components in the buffer for electrophoresis changes with time. By performing the analysis, the gel is not prepared each time the analysis is performed, and the separation and analysis of DNA of a wide range of sizes are performed continuously with good reproducibility.

[Means and Actions for Solving Problems] As a result of intensive studies to solve the above problems, the present invention has been achieved. That is, the present invention does not fill an agarose-based or polyacrylamide-based gel into the capillaries when performing separation and analysis of DNA or the like using capillary electrophoresis, and does not gelate the agarose-based gel in the electrophoresis buffer. It is a capillary electrophoresis method characterized by containing a polymer or a cellulosic polymer which is not gelled, and changing its concentration with time.

The sample for capillary electrophoresis is not particularly limited, but a solution containing a polymer such as nucleic acid or protein is most suitable for separation analysis.

In the present invention, so-called low melting point agarose having a low gelling temperature is preferable as the agarose-based polymer added to the electrophoresis buffer, but any agarose-based polymer that does not gel during use may be used. You can Hydroxymethyl cellulose is preferable as the cellulosic polymer, but other polymers can be used.

Further, it is preferable to add 0.01 to 0.5% of a surfactant such as SDS (sodium dodecyl sulfate) to the buffer solution for electrophoresis, but it is not necessary to add it.

As the electrophoresis buffer, for example, one containing 0.1 M tris (hydroxymethyl) aminomethane and boric acid as a buffer is used, but various buffers may be used depending on the sample to be separated and analyzed. it can.

The material of the capillaries is preferably fused silica, but is not limited to this.

The inner diameter of the capillary is preferably 10 to 200 μm, but not limited to this. The length of the capillary is preferably 50 mm or more, but is not limited to this.

Further, the power source preferably has a maximum output voltage of about 30 kV, but may be smaller than this or may be larger than this. Further, the electric current is preferably direct current, but may be generated in a pulse shape and is not limited to these.

As the detector, for example, a UV detector or a fluorescence detector is preferable, but an electrochemical detector or the like may also be used.
It is not limited to these.

The recorder preferably has a data processing function such as retention time, peak height, and peak area calculation, but is not limited to this.

Further, it is preferable that the control unit can control a plurality of pumps.

The sample is introduced into the capillary filled with the electrophoresis buffer solution from the ends, and both ends of the capillary are immersed in separate electrode tanks containing the electrophoresis buffer solution. A Pt electrode is immersed in each of these two electrode tanks, and a voltage is applied to both electrodes.
By applying a voltage across the capillary, a flow occurs in the electrophoresis buffer solution inside the capillary, and the components of the eluted sample are detected by the above detector. The electrical signal from the detector is transmitted to the recorder for processing. Further, during the electrophoresis, the composition of the buffer solution for electrophoresis in the electrode tank on the + electrode side changes with time by the pump controlled by the controller.

[Examples] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Separation and analysis of a mixture of restriction enzyme-treated fragments of DNA 1. Preparation of electrophoresis buffer containing low-melting point agarose Add 0.5 g of low-melting point agarose to 80 ml of distilled water, stir well, heat and dissolve to release. After cooling, 1.21 g of tris (hydroxymethyl) aminomethane, 93 mg of disodium ethylenediaminetetraacetate and 20 mg of sodium dodecyl sulfate were dissolved therein. Boric acid is added to this, and the pH
Was adjusted to 8.1 and distilled water was added to make exactly 100 ml.

2. Preparation of electrophoresis buffer containing no low melting point agarose 1.21 g of tris (hydroxymethyl) aminomethane, 93 mg of disodium ethylenediaminetetraacetate and 20 mg of sodium dodecyl sulfate were dissolved in 90 ml of distilled water.

Boric acid was further added to this to adjust the pH to 8.1, and distilled water was added to make exactly 100 ml.

3. Capillary electrophoresis of a mixture of DNA fragments treated with restriction enzymes Capillary electrophoresis was performed using the system shown in FIG. That is, the fluorescence detector (1) is RF-540 type (manufactured by Shimadzu Corporation, excitation wavelength is 300 nm, detection wavelength is set to 590 nm), and the high voltage power source (2) is HER-30P0.16-SI type (manufactured by Matsusada Precision Devices). ), The recorder (3) is a C-R4A type (manufactured by Shimadzu Corporation), and the electrode (4) is a Pt wire (0.5 mmφ-30 m).
m), LC-6A type (manufactured by Shimadzu) as the pump (8), and SCL-6A type (manufactured by Shimadzu) as the control unit (11).

As the capillary (12), a fused silica capillary having an inner diameter of 75 μm manufactured by Scientific Glass Engineering was used. The total length of the capillary was 450 mm, and the coating was peeled off with a width of 2 mm from 300 mm from the + side and attached to the fluorescence detector. The capillary was filled with the above-mentioned electrophoresis buffer solution at the time of use, and both ends were immersed in the + electrode side electrode tank 6 and the − electrode side electrode tank 5 respectively containing the electrophoresis buffer solution. At this time, the heights of the liquid surfaces of the buffer solutions in the two electrode tanks were adjusted to be the same.

Two kinds of commercially available products were used as the mixture of the restriction enzyme-treated fragments of the sample DNA (Nippon Gene marker 5 (φx174 / HincI
I digest 79-1057 base pairs, 0.5 μg / ml) and Nippon Gene marker 1 (λ / HindIII digest 0.13-23.13k)
Base pairs, 0.5 μg / ml)) were mixed and used. The sample was introduced into the capillary by pulling up the end on the + electrode side of the capillary from the + side electrode tank and immersing it in the sample solution for 10 seconds.
At this time, the height of the liquid surface of the sample was adjusted to be 50 mm higher than the liquid surface of the buffer solution in the electrode tank.

After introducing the sample into the capillary, return the end of the capillary to the electrode tank and apply a DC voltage of 7.5 kV to both ends of the capillary. Also, the low melting point agarose concentration in the + electrode side electrode tank is as shown in Fig. 2. The pump was controlled so that
The current value is 12 to 15 μA, and the buffer solution flows from the positive electrode side to the negative electrode side in the capillary, which is a sample.
Each restriction enzyme treated fragment of DNA was separated, eluted and detected with a fluorescence detector.

The results are shown in FIG. From these results, it can be seen that the method of the present invention successfully separated and detected a mixture of restriction enzyme-treated fragments of DNA in a wide range of sizes.

In FIG. 1, 7 is a drain, 9 is a mixing block, and 10 is an electrophoresis buffer reservoir.

[Effects of the Invention] According to the method of the present invention, since the electrophoresis buffer having the optimum composition for separation is constantly supplied into the capillary, the separation analysis can be performed with good reproducibility.

[Brief description of drawings]

FIG. 1 is a diagram showing an apparatus configuration for carrying out the method of the present invention, FIG. 2 is a diagram showing changes in the low melting point agarose concentration in a buffer solution, and FIG. 3 is a diagram showing separated and detected data. is there. 1 ... Fluorescence detector, 5 ...- Pole side electrode tank, 6 ... + Pole side electrode tank, 8 ... Pump, 10 ... Buffer solution reservoir, 11 ...
… Pump controller, 12… Capillary

Claims (1)

[Claims] 1. A capillary electrophoresis method, characterized in that a non-gelling agarose polymer or non-gelling cellulose polymer is contained in a buffer for electrophoresis, and the concentration thereof is changed with time. .