KR970010972B1 - Post electrophoresis capillary scanning apparatus - Google Patents

Abstract

There is provided a scan-detecting device which is useful for sequencing technique of DNA basic order and which is used after capillary tube gel electrophoresis using laser. The device is comprised of a light source means(20) for providing light of different wave each other for investigation through sample, a means for moving capillary tube(10) for sample to be scanned by light, a means(30) for collimating light which is investigated through sample and generated, a means(40) for selecting interactive wave with sample from the collimated light, and a means(50) for detecting light of the selected wave.

Description

Device for scanning and analyzing the sample developed by capillary

1 is an overall explanatory diagram showing a scan-detection apparatus after capillary electrophoresis deployment using laser induced fluorescence using an independent four-channel detector.

FIG. 2 is a schematic diagram illustrating a post-capillary gel electrophoretic post-scan-detection device for independent four-channel laser induced fluorescence detection using a motor and roller to draw capillaries.

3 is a schematic diagram of a device used to place a solution inside a capillary tube while easily handling the capillary tube from which the coating is removed.

4 is a schematic representation of a capillary holder wound around a long capillary tube for ease of handling.

FIG. 5 is an electrophoresis graph obtained by attaching a fluorescent label to a pGEM-3zf (+) DNA sample and developing and separating the product by capillary gel electrophoresis, followed by a capillary scan-detection apparatus according to the present invention.

* Explanation of symbols for main parts of the drawings

10 capillary 20 light source

30: optical fiber 40: optical filter

50: photomultiplier tube 60: chart recorder

The present invention relates to a laser-capillary post-scan-detection apparatus useful as a sequencing technique for deoxyribonucleic acid (DNA).

DNA sequencing is a very important research tool in life sciences. Currently, the commonly used method is polyacrylamide gel electrophresis of isotopically substituted sequencing reaction products, which are separated according to molecular weight, and each component is detected by radioradiography. To determine the DNA sequence. This classic method is still in use, but the labor force is enormous. In the late 1980s, a method of attaching a fluorescent label instead of an isotope and detecting it with a laser was developed and commercialized by US Applied Biosystems or Du Pont. All of these methods use slow gel plate electrophoresis to separate sequential conjugation reaction products. In order to increase the speed, Joule heating occurs when the strength of the electric field is increased. Therefore, the strength of the electric field can be raised above 50 V / cm.

Therefore, many studies have recently been conducted to use capillary gel electrophoresis (CGE) for DNA sequencing due to fast capillary tubes. However, filling the capillary gel with a large area-to-volume ratio allows for good heat dissipation, so that a very placed electric field, such as 400V / cm, can be applied. However, it is not possible to separate and detect several samples at the same time.

In other words, CGE has a much higher separation rate than plate gel electrophoresis, which is a useful separation method for DND sequencing.However, in general CGE, sequencing reaction products are developed using a single capillary tube to detect and separate several samples. The overall DNA sequencing rate is not much faster than automated plate gel electrophoresis systems that simultaneously detect and detect electrophoresis.

The present inventors have developed a method of determining DNA sequences by separating DNA samples into CGE and then scanning the capillaries with a laser in order to fully take advantage of the fast separation rate of CGE. Scanning time is a few minutes, which is much shorter than the CGE time. Therefore, several samples can be simultaneously CGE and then one laser is scanned by laser to detect laser induced fluorescence. By determining the DNA sequence, the overall sequencing rate is several orders of magnitude faster.

It is therefore an object of the present invention to provide an apparatus for determining DNA sequences faster than automated plate gel electrophoresis systems by determining samples, particularly DNA sequences, at an extremely fast rate.

The present invention provides a device for analyzing a sample by irradiating light (25) to a sample developed by a capillary, comprising: light source means (20) for providing light of different wavelengths for being irradiated onto the sample; Means for moving the capillary tube such that the sample is scanned by light; Means (30) for irradiating light onto the sample to focus the generated light; Means (40) for selecting a wavelength that interacts with a sample in the focused light; And means (50) for detecting light of the selected wavelength.

Figure 1 shows an overall explanatory diagram of an apparatus of the present invention for scanning and detecting CGE after developing a sample with CGE for DNA sequencing. As shown in FIG. 1, the laser beam from the argon ion laser is pulled out by capturing the capillary tube by operating the motor 16 to the capillary tube which has been developed and separated from the sequencing reaction product by CGE (see FIG. 2). Focus the beam on the capillary with a lens, and select only the wavelength from the fluorescent label of one base among the fluorescence from the other end of the fiber in the direction perpendicular to the laser beam using various optical filters 40 The photomultiplier tube (50) is detected and the electrical signal from the photomultiplier tube is recorded on a strip-chart recorder (60).

In the present invention, the polyimide coating of the capillary is peeled off before filling the polyacrylamide gel in the capillary, in order to prevent absorption from occurring when the laser light is incident into the capillary and the induced fluorescence is collected. In the present invention, when the electrophoresis is performed, it is preferable to use a capillary tube long enough so that any component separated after the sample is separated into the component does not exit the capillary tube and remains in the capillary tube without leaving the capillary tube.

Fluorescent labeling used for DNA sequencing is different for each base, and accordingly, two wavelengths of excitation light sources are required: 488 nm and 514 nm. 488nm excites fluorescent labels corresponding to adenine and cytosine, and 514nm excites fluorescent labels corresponding to guanine and thymine.

Acquire the 488nm and 514mn light of the argon-ion laser's multicolored visible light by prism, and focus the light at two different points on the capillary. The space around this part is a liquid with the same or similar refractive index as the capillary material. It is preferable to fill the medium of.

At the point where the light of one skin is focused, the two optical fibers are located near the capillary, facing each other in a direction perpendicular to the excitation laser beam.

At the point where light of one wavelength is focused, the two optical fibers are located near the capillary, facing each other in a direction perpendicular to the excitation laser beam.

Behind the light exit of each optical fiber is a cylindrical glass rod 41 for condensing, optical filters for selecting a fluorescence wavelength of a label corresponding to a base to be detected, and a photomultiplier tube 50. As an example of the optical filter, the long-pass filter 42, the color glass filter 43, and the band-pass filter 44 may be configured to be continuously arranged.

In addition to scanning by pulling the capillary tube as shown in FIG. 2, the capillary-filled gel may be scanned and detected while pushing the capillary-filled gel under pressure while the capillary tube and the detector are fixed. The post-GEGE scan-detector is available for all DNA assays using capillary electrophoresis, as well as for capillary gel separation. Alternatively, a capillary filled with another polymer solution may be used instead of the gel. The post-separation scan-detection device has the advantage of shortening the overall analysis time when several samples are to be analyzed, and also reducing the actual use time of the expensive laser when using the laser as a light source.

As described above, the analysis apparatus according to the present invention has been described based on DNA sequencing. In addition, the sample developed by the capillary tube by capillary chromatography or the like is analyzed by the apparatus of the present invention by selecting a light source having a suitable wavelength interacting with the sample. can do.

Next, embodiments of the present invention will be presented, but the scope of the present invention is not limited thereto.

Example 1

Separation of DNA sequencing reaction products by filling the capillary with gel and electrophoresis (see FIGS. 3 and 4)

In order to scan the entire capillary 10, only a suitable amount of the polyimide coating of the capillary tube is left at both ends of the capillary tube and the coating between the portions is removed. The capillary is placed in concentrated sulfuric acid and heated to remove the polyimide coating 71 on the capillary outer wall. The capillary 10 having the coating removed is placed in a tube 70 made of polypropylene resin attached to a polyethylene tube 73 at both ends for easy handling. To put the solution 72 inside the capillary, one end is immersed in the solution and the other is depressurized by applying a vacuum 75 to the flask 74 (see FIG. 3). It is difficult to use a long capillary tube with a straight tube. To solve this problem, a capillary tube wound around a holder such as a fourth degree can be easily used for several meters of long capillary tube.

The method of filling the polyacrylamide gel in the capillary is as follows. 0.1N NaOH, 0.1N HCL, and 0.1N NaOH solution were flowed into the capillary (100 μm in inner diameter and 200 μm in outer diameter) in order to wash the inside of the capillary, followed by rinsing with distilled water and methanol. Fill the capillary with a 1: 1 solution of methanol and methacryloxypropyl trimethoxysilane (MAPS) in the washed capillary and leave it overnight so that the inner wall of the capillary is completely replaced with MAPS. The buffer is ph 8.3, 90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA. 7M urea is added to a 9% acrylamide solution made with this buffer as a solvent, followed by degassing. 10% ammonium persulfater and 10% tetramethylenediamine (N, N) are added to the solution. , N ', N'-tetramethylendiamine (TEMED) solution was added to a polyacrylamide gel capillary tube made up to 0.03% of the total solution, respectively, and then placed in a refrigerator (6 ° C.) overnight to harden the gel.

DNA samples are made by enzymatic methods using fluorescently labeled primers (manufactured by Primer Applied Biosystems) to generate induced fluorescence with laser light instead of radioisotopes.

Fluorescent labels with different fluorescence wavelengths are attached to each of adenine, cytosine, guanine, and thymine.

After injecting the DNA sample into the gel-filled capillary for 30 seconds, the DNA sample was subjected to electrophoresis by applying a 200-300V / cm voltage.

Example 2

Fabrication of the Scan-Detection Device after CGE Deployment Using Laser-Induced Fluorescence (see FIGS. 1 and 2)

Fluorescent labels used to determine the DNA sequencing use different ones for each base, and accordingly, two wavelengths of the excitation light source need 488 nm and 514 nm, respectively. Therefore, 488 nm and 514 nm light are separated by the prism 21, the grating, and the slit 22 by the multi-color visible light of the argon ion laser 20, which is a light source (see FIG. 1), or the 488 nm wavelength. The light is split into a beam splitter, and one side is incident to the capillary as it is, and the other is sent to a dye laser to obtain 514 nm, which is then incident on the capillary. This light beam can change course to the mirror 23 as needed. 488 nm of laser light 25 irradiated to the capillary excites fluorescent labels corresponding to adenine and cytosine, and 514 nm filters fluorescent labels corresponding to guanine and tamine. Four optical fibers 30 are used to condense fluorescence so that two optical fibers 30 face each other at right angles to two laser lights. It is preferable to fill a liquid having a refractive index similar to that of the capillary material in the space around the portion where the capillary laser beam 25 is collected. Light from each optical fiber is passed through a color glass filter, a long wave-pass filter, and a bandpass filter in order to remove scattered light and to selectively pass only the fluorescence of a desired area. After detection, it is detected by a photomultiplier tube (PMT). The electrical signal from the photomultiplier tube 50 is passed through a suitable amplifier and recorded on a chart recorder or stored in the on-computer 60 using an analog-to-digital converter 60.

2 is a four-channel laser induced fluorescence detection device for detecting laser induced fluorescence of DNA samples separated by capillary electrophoresis. The electrophoretic capillary (10) was inserted between the two rollers (13) through a polyetherether ketone (PEEK, 12) tube having an outer diameter of 1/16 "and an inner diameter of 0.01" in the capillary holder (11). Then turn the roller at a constant speed to pull out the capillary. The roll furnace 13 is driven by, for example, a power source 15, a motor and gear box 16, a rubber band 14, and the like. The laser-induced fluorescence emitted by the laser light 25 irradiated to the capillary passes through four optical fibers 30 in the perpendicular direction, and then independently passes through the four wavelength selecting means 40 to the photomultiplier tube 50. do.

Example 3

DNA sequencing using a scan-detector post-CGE deployment using laser-induced fluorescence (see Figure 5)

FIG. 5 is an example graph showing the results of laser induced fluorescence of guanine out of four bases of DNA using a photomultiplier tube and recording with a chart recorder using a post-CGE scan-detection device, wherein the DNA sample is pGEM-3zf. (+). The capillary was 60 cm in length, and the sample was injected at 15 kV for 30 seconds and electrophoresed at 15 kV for 45 minutes. A voltage of 1070 V was applied to the photocathode of the photomultiplier tube.

The voltage applied to the motor was 3.5V and the pulling rate of the capillary was 1mm / sec. The cheap sequence was determined up to 215 bases of guanine, and the time required to scan the capillary was 8 minutes.

In order to record electrophorograms of four bases simultaneously in four colors on a graph and to make scanning faster, the signal from the photomultiplier must be computerized using an analog digital converter. do. Current scan speed is limited by the recording speed of the chart recorder. When the computer processes the signal, the scan speed can be reduced to less than 2 minutes. Then, when the optical fiber holder of FIG. 2 is filled with a liquid (for example, glycerol) having a refractive index almost similar to that of molten silica, the material from which the capillary and the optical fiber are made, when the laser beam enters the capillary or the laser-induced fluorescence exits the capillary Since the scattering that occurs when entering the circuit can be almost eliminated, the signal-to-nosiser ratio of the detection signal can be reduced, further improving the sensitivity of the detection device. The length of the capillary tube used to obtain the electrophoretic graph of FIG. 5 was 60 cm. If a longer capillary tube is used, the number of smoke that can be separated, detected and determined by this method will be much higher.

Although preferred embodiments of the present invention have been described above, the scope of the present invention is not limited thereto, and it should be understood that substitutions, changes, or modifications apparent to those skilled in the art are within the scope of the present invention.

Claims (18)

An apparatus for analyzing a sample by irradiating light (25) to the sample developed by the capillary, the apparatus comprising: light source means (20) for providing light of different wavelengths to be directed to the sample; Means for moving the capillary tube 10 such that the sample is scanned by light; Means (30) for concentrating light generated by irradiating light onto a sample; Means (40) for selecting a wavelength that interacts with a sample in the focused light; And means (50) for detecting light of the selected wavelength. The device of claim 1, wherein the capillary development means is capillary gel electrophoresis (CGE) or capillary chromatography. The apparatus according to claim 1, wherein the sample is a DNA-containing sample, and the focusing means is four channels. The device according to claim 3, wherein fluorescent labels having different fluorescence wavelengths are attached to each of adenine, guanine, cytosine and thymine. The device of claim 1, wherein the sample is a peptide or an amino acid. An apparatus according to claim 1, wherein said light source means is connected to a laser generator or a multicolor continuous light source. 7. The apparatus of claim 6, wherein the laser generator is an argon ion laser generator. 2. An apparatus according to claim 1, wherein the light of different wavelengths (25) have wavelengths of 514 nm and 488 nm, respectively. The device of claim 1, wherein the capillary is filled with a medium having the same or similar refractive index as the capillary material in a space around the light-collecting portion. A device according to claim 1, wherein the device for focusing fluorescence is an optical fiber (30). 11. An apparatus according to claim 10, characterized in that the two optical fibers (30) are located facing each other in a direction perpendicular to the light irradiated at the point where the light is irradiated to the sample. 2. Device according to claim 1, characterized in that the capillary moving means pulls the capillary (10) by the driving of the roller (13) and the motor (16). The device of claim 12, wherein the moving speed of the capillary tube is at least 1 mm / sec. 13. Device according to claim 12, characterized in that the capillary is wound around the capillary holder (76) for use. 2. Device according to claim 1, characterized in that the capillary tube (10) has its coating removed on its outer wall. The device according to claim 2, wherein the capillary is filled with a polymer solution to develop a sample. 2. Device according to claim 1, characterized in that the device for selecting the wavelength is an optical filter (40). 18. The apparatus according to claim 17, wherein the optical filter is continuously composed of a long pass filter (42), a color glass filter (43) and a band pass filter (44).