JP2722752B2 - DNA detection method - Google Patents

Description

The present invention relates to a method for detecting DNA.

More specifically, the present invention relates to a method for detecting DNA using gene amplification technology and capillary electrophoresis.

[Prior Art] In recent years, with the development of genetic engineering, methods for detecting DNA have been developed and tried. For example, DNA probe methods using oligonucleotides, hybridization methods have been attempted, and DNA has been separated by electrophoresis using an agarose gel containing ethidium bromide, and UV
The fluorescence generated by irradiating the DNA
Has been detected.

Applications utilizing the detection of DNA as described above have been spread to various fields, for example, detection of transfectants into which genes have been introduced, diagnosis of genetic diseases, and the like. It is.

Conventionally, in the microbial test, in the diagnosis of infectious disease or the bacterial test of food, first, bacteria and the like present in the sample are enriched and cultured under aerobic and anaerobic conditions, and the colonies are observed. The bacteria are identified by separating and culturing them, observing the colonies, and examining various properties of the bacteria. Alternatively, by eliminating the enrichment culture step and separating and culturing bacteria and the like present in the sample using a selective medium under aerobic and anaerobic conditions, observing the colonies, and examining the various properties of the bacteria, (Clinical Laboratory Techniques, Vol. 7, Microbiology, edited by Nozomu Sakai, published in 1985 by the Medical College).

In recent years, even in such a microorganism test, a DNA probe method or a hybridization method using an oligonucleotide has been attempted, and practical use of the microorganism test by detecting DNA is expected.

DNA is a gene amplification method using an oligonucleotide as a primer (Polymerase Chain Reaction, hereinafter abbreviated as PCR; Saiki et al., Science, 230, 1350 (1985)).
A method for amplification is known, and a method using the amplified DNA has been attempted for DNA detection.

[Problems to be Solved by the Invention] Among DNA detection methods, particularly in microbial tests and the like, hybridization is performed on a membrane or another support with a probe labeled with an oligonucleotide, and the labeled probe is used. In fact, it is difficult for the detection method to obtain sufficient detection sensitivity and selectivity.

In addition, in the method of amplifying DNA derived from a microorganism obtained from a sample by PCR and detecting the amplified DNA by agarose gel electrophoresis, it takes time to prepare a gel, the gel is difficult to handle, and the sample is applied to the gel. There are disadvantages, such as taking time and effort, and easy to understand but not quantitative. Therefore, there is a problem when automation or equipment is to be used, particularly in fields requiring quick, simple and high sensitivity, such as microbial testing. Under such circumstances, development of a rapid, simple, and high detection sensitivity DNA detection method is expected.

An object of the present invention is exactly at this point, and it is an object of the present invention to provide a method for quickly and easily detecting a DNA amplified by using a PCR method.

Specifically, the number of theoretical plates is high, sufficient separation can be obtained, the analysis time is short, the sample processing capacity per time is high, the sample can be easily injected, the number of samples may be small, and gel preparation is not required. An object of the present invention is to provide a method for detecting DNA which has advantages such as low running cost and good quantitative and reproducibility.

[Means and Actions for Solving the Problems] As a result of intensive studies to achieve the above object, the present inventor first used a PCR method to extract a part of DNA in a sample when performing a microorganism test or the like. It has been found that DNA can be detected quickly, easily and with high sensitivity by detecting the amplified DNA using capillary electrophoresis, and further studies have led to the completion of the present invention. Was.

Here, the DNA detected in the present invention may be of any origin depending on the purpose of detecting the DNA. For example,
In microbial testing, DNA derived from microorganisms is used,
When used as a method for detecting a transformant into which a gene has been introduced, the DNA of the introduced foreign gene is used. In the case of a genetic disease, DNA having a partial mutation in the nucleotide sequence is used.

In the present invention, a known method (Biochemical Experiment Course 2,
Nucleic acid chemistry I, 41-54 (1975), edited by The Biochemical Society of Japan, published by Tokyo Kagaku Dojin), first, DNA is extracted from the target microorganisms in the sample, and the DNA is subjected to PCR (Polymerase).
Following the Chain Reaction method, Saiki et al., Science, 230, 1350 (1
985)). PC containing this amplified DNA
After removing the upper layer mineral oil, the R reaction solution is directly analyzed by capillary electrophoresis.

Capillary electrophoresis is performed by the following method, but the analysis conditions and operation method are not limited thereto.

In the capillary electrophoresis used in the present invention, electrophoresis is performed by adding a non-gelled polymer to an electrophoresis buffer, but any polymer that does not gel during use can be used. . For example,
So-called low-melting point agarose and starch having a low gelation temperature can be mentioned. In the present invention, low-melting point agarose is particularly used.

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

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

The material of the capillary is preferably, but not limited to, fused silica. For example, a Teflon tube or the like may be used. The inner diameter of the capillary is preferably from 10 to 200 μm, particularly preferably from 50 to 100 μm, but is not limited thereto. The length of the capillary is usually 30 to 50 cm, but is not limited thereto.

Further, the power supply is preferably a power supply having a maximum output voltage of about 30 kV, but may be a power supply having a smaller output voltage or a power supply having a higher output voltage. The current is preferably a direct current, but may be a pulse-like current, and is not limited thereto.

The detector is preferably, for example, a UV detector or a fluorescence detector, but may be an electrochemical detector or the like, and is not limited thereto.

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

A sample is introduced from the end into the capillary filled with the above-mentioned electrophoresis buffer containing the polymer and the like, and both ends of the capillary are immersed in separate electrode tanks containing the electrophoresis buffer containing the polymer and the like. . A Pt electrode is immersed in each of these two electrode tanks, and a voltage is applied to both electrodes. By applying a voltage to both ends of the capillary, a flow occurs in the buffer for electrophoresis containing the polymer inside the capillary,
The components of the eluted sample are detected by the detector described above. The electrical signals from the detector are transmitted to a recorder and processed there.

[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.

Detection of Salmonella 1. Preparation of sample Salmonella is inoculated into an appropriate enrichment medium using Salmonella typhimurium , cultured overnight at 37 ° C under aerobic conditions, and cells are collected by centrifugation from 1.5 ml of the medium. did. After washing once with 10 mM Tris-HCl buffer (pH 7.5),
A solution of lysozyme dissolved at 1 mg / ml in the same buffer
The suspension was suspended in 0.5 ml and lysed at 37 ° C. for 10 minutes. The same volume of phenol saturated with the buffer was added to the lysate, and the mixture was stirred well. After centrifugation, the upper layer solution was recovered and subjected to ethanol precipitation to precipitate nucleic acid components. The precipitate was dissolved in 1 ml of the above buffer solution, and this was used as a sample.

2. Synthesis of primers As primers for specifically amplifying Salmonella DNA, oligonucleotides having the following base sequences (Japanese Patent Application
1-185683) was chemically synthesized.

(5 ′) d-GGCGAGCAGTTTGTCTGTC (3 ′) (5 ′) d-GTTTCGCCTGGCTGATACG (3 ′) Chemical synthesis was performed by the triester method using Shimadzu DNA synthesizer NS-1. Purification of synthesized oligonucleotides
This was performed using a C18 reverse phase column.

3. PCR Use 3 µl of the sample solution and add sterile distilled water to 16.05 µl.
l, 3 μl of 10 × reaction buffer, 4.8 μl of dNTP solution, 1.5 μl of each primer and heat-resistant DNA polymerase
0.15 µl was added to prepare 30 µl of a reaction solution. 50 μl of mineral oil (manufactured by SIGMA) is added to the container containing the reaction solution.
l and layer on the reaction solution. The content of each added liquid is shown below.

10 × reaction buffer; 500 mM KCl, 100 mM Tris-HCl (pH 8.
3), 15 mM Mgcl ₂ , 0.1% (w / v) gelatin dNTP solution; a mixture of dATP, dCTP, dGTP, and dTTP, each having a final concentration of 1.25 mM primer; OD
unit / ml) Thermostable DNA polymerase; Taq DNA polymerase (5 unit /
ml; manufactured by Perkin Elmer Cetus) The reaction conditions are as follows.

Thermal denaturation; 94 ° C for 1 minute Annealing; 37 ° C for 1 minute Polymerization reaction; 60 ° C for 1 minute The process from heat denaturation to annealing and annealing to polymerization is defined as one cycle (time required: 5.7 minutes). 4 hours). These operations are performed by Perkin
This was performed by programming the above reaction conditions in a DNA Thermal Cycler manufactured by Elmer Cetus.

4. Detection (capillary electrophoresis) (4-1) Preparation of buffer for electrophoresis Add 0.5 g of low melting point agarose to 80 ml of distilled water, stir well, heat and dissolve, allow to cool, 1.21 g of tris (hydroxymethyl) aminomethane, 93 mg of disodium ethylenediaminetetraacetate, 20 mg of sodium dodecyl sulfate and 100 μg of ethidium bromide were dissolved. To this, boric acid was further added to adjust the pH to 8.1, and distilled water was added to make exactly 100 ml.

(4-2) Capillary Electrophoresis of PCR Reaction Solution Containing Amplified Salmonella DNA 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 300 nm, detection wavelength 590 nm), and the high voltage power supply (2) is HER-30P 0.16-SI type (Matsusada Precision Devices). , Recorder (3) is C-R4A type (manufactured by Shimadzu Corporation), electrode (4) is Pt wire (0.5mmφ-30m)
m), a 1.5 ml sampling tube was used for the electrode tank (5).

Capillary (6) is Scientific Glass Engineering
A fused silica capillary with an inner diameter of 75 μm was used. The total length of the capillary was 450 mm, and the coating was peeled off at a width of 2 mm from a position 300 mm from the + pole side, and attached to a fluorescence detector. Fill the capillary with an electrophoresis buffer containing the low melting point agarose at the time of use,
Both ends were immersed in a positive electrode tank and a negative electrode tank containing an electrophoresis buffer containing low-melting-point agarose, respectively. At this time, the heights of the buffer solutions in the two electrode tanks were adjusted to be the same.

The sample was introduced into the capillary by pulling up the positive end of the capillary from the positive electrode tank and immersing it in the sample solution for 10 seconds. At this time, the liquid level of the sample was adjusted so as to be 50 mm higher than the liquid level of the buffer solution in the electrode tank.

After the sample was introduced into the capillary, the end of the capillary was returned to the electrode tank, and a DC voltage of 7.5 kV was applied to both ends of the capillary. The current value was 12 to 15 μA, and a buffer solution flowed from the positive electrode side to the negative electrode side in the capillary, DNA was separated, eluted, and detected by the fluorescence detector. Salmonella DNA amplified by the PCR reaction was detected as a sharp peak at the expected elution time. The result is shown in FIG.

[Effects of the Invention] According to the method of the present invention, DNA is separated and detected by capillary electrophoresis using an electrophoresis buffer containing low-melting point agarose, so that the separation environment in the capillary becomes homogeneous. , Amplified DNA after PCR reaction
Can be analyzed quickly and easily with high reproducibility and sufficient resolution without preparing a gel, and the sample volume required for capillary electrophoresis can be reduced.
The PCR reaction system can be made smaller and running costs can be kept low.

[Brief description of the drawings]

FIG. 1 shows a system for capillary electrophoresis, and FIG. 2 shows detection of amplified Salmonella DNA by a fluorescence detector. 1 Fluorescence detector 2 High voltage power supply 3 Recorder 4 Electrode 5 Electrode tank 6 Capillary

Claims (1)

(57) [Claims] 1. A method for detecting DNA, wherein the DNA amplified by the gene amplification method is detected by capillary electrophoresis using an electrophoresis buffer containing low-melting-point agarose.