WO2006111082A1

WO2006111082A1 - A device for real-time and continuous monitoring gel electrophoresis and method thereof

Info

Publication number: WO2006111082A1
Application number: PCT/CN2006/000715
Authority: WO
Inventors: Yunkang Liu
Original assignee: Yunkang Liu
Priority date: 2005-04-18
Filing date: 2006-04-18
Publication date: 2006-10-26
Also published as: CN1854716A; CN1854716B

Abstract

An electrophoretic analysis device, which includes a transmission analysis plate and narrow-spectrum light sources or pure spectrum light sources disposed on the two sides of the transmission analysis plate. Said light sources are inclined by a horizontal angle with respect to the gel plate. A method for real-time and continuous monitoring gel electrophoresis uses the said device and uses the pure spectrum that can match the added fluorescent dye. Said pure spectrum is inclined by a horizontal angle with respect to the gel plate. Said fluorescent dye is excited by the pure spectrum. So the position of the samples in the gel plate can be shown such that the samples can be monitored real-time and continually.

Description

Electrophoretic separation and analysis device integrated with convertible light source and use thereof

The present invention relates to a gel electrophoresis separation and analysis device for a naturally fluorescent or charged substance bound to a fluorescent label. In particular, the present invention relates to a highly integrated multi-purpose electrophoretic separation and analysis device that can change the wavelength of a light source. Background technique

Fluorescence and Fluorescent Matter _ ; When light of a certain wavelength (incident light) is irradiated to certain substances, these substances emit light (emitting light) different from the wavelength and intensity of the incident light. When the incident light stops, the light emitted by the irradiated material also disappears. The light emitted by the substance to be irradiated is called fluorescence. A substance capable of emitting fluorescence is called a fluorescent substance. N. Monardes first recorded fluorescence in 1575. He observed a very cute sky blue fluorescence in an aqueous solution of a slice of wood called "LignumNephriticum". In the 17th century, famous scientists such as Boyle (1626-1691) and Newton (1644-1727) observed fluorescence again and gave a more detailed description. When Stokes examined the fluorescence of quinine and chlorophyll in 1852, it was observed with a spectrometer that the wavelength of the fluorescence was slightly longer than the wavelength of the incident light. It was found that this phenomenon is that these substances re-emit light of different wavelengths after absorbing light energy. Instead of being caused by the diffusing action of light, the introduction of fluorescence is the concept of light emission, and in 1864, fluorescence was first proposed as an analytical tool. In 1867, GoppdsrMer) performed the first fluorescence analysis work in history, using the fluorescence of aluminum-mulberry complexes for the determination of aluminum. In 1880, Liebeman proposed the earliest rule of thumb for the relationship between fluorescence and chemical structure. By the end of the 19th century, more than 600 fluorescent compounds including fluorescein, eosin, and polycyclic aromatic hydrocarbons were known. Since the 20th century, more in-depth research has been carried out on the phenomenon of fluorescence.

The development of fluorescence analysis methods is inseparable from the development of instrument applications. Before the 19th century, the observation of fluorescence was carried out by the naked eye. It was not until 1928 that the first photoelectric fluorometer was proposed by Jette and West. The sensitivity of early optoelectronic fluorometers was limited. After the invention of photomultiplier tubes by Zworykin and Rajchman in 1939, it was a very important stage in terms of increasing sensitivity and allowing the use of higher resolution monochromators. In 1943, Dutton and Bailey proposed a manual calibration procedure for fluorescence spectroscopy. In 1948, Studer introduced the first automatic spectroscopy correction device. It was not until 1952 that commercial calibration spectroscopy instruments appeared. In the past decade or so, under the influence of the rapid development of other disciplines, with the new achievements of lasers, microprocessors and electronics, and the discovery of new fluorescent materials, the synthesis, such as synchronous fluorescence measurement, derivative fluorescence measurement, Some new methods for fluorescence analysis, such as inter-resolution fluorescence measurement, phase-resolved fluorescence measurement, fluorescence polarization measurement, fluorescence immunoassay, cryogenic fluorescence measurement, solid surface fluorescence measurement, fluorescence reaction rate method, three-dimensional fluorescence spectroscopy, and fluorescent fiber chemical sensor The development of new technologies, and correspondingly accelerated the advent of a variety of new types of fluorescence analysis instruments, so that fluorescence analysis methods continue to develop in the direction of high efficiency, trace, micro and automation, the sensitivity, accuracy and choice of methods Increasingly, the application of the method has been greatly expanded, covering various fields such as industry, agriculture, medical and health, environmental protection, public security intelligence and scientific research. Today, fluorescence analysis has evolved into an important and effective means of spectrochemical analysis.

^ ^^ [5,6] _; Charged particles move toward an electrode of opposite polarity under the action of an electric field, called electrophoresis. The phenomenon of charged particles moving in an electric field was first observed by Professor Svedberg, Department of Physical Chemistry, Uppsal University, Sweden. In 1937, Swedish scientist Tiselius designed the world's first electrophoresis-transition boundary electrophoresis method. However, due to the density change caused by the free solution heated by the "migration electrophoresis" electrophoresis, the convection is not high and the electrophoresis instrument is expensive, which is not promoted. In the past 50 years, the electrophoresis system has been improved and the filter paper, cellulose acetate film, starch, agarose, etc. have been used as supporting media, and electrophoresis technology has been promoted and applied. In the past 60 years, polyacrylamide gel was found as a supporting medium and SDS-polyacrylamide electrophoresis, isoelectric point electrophoresis, two-dimensional electrophoresis and blot transfer electrophoresis were developed. These technologies have the advantages of simple equipment, convenient operation, and high resolution. Electrophoresis technology has become an essential analytical tool in biochemistry, immunology, molecular biology, and closely related fields of medicine, agriculture, forestry, animal husbandry, fish, and pharmaceuticals.

Any substance that moves to a certain electrode in an electric field due to its own dissociation or adsorption of other charged particles on the surface. The charged particles may be small ions, biological creatures, proteins, nucleic acids, viral particles, organelles, and the like. Different charged particles have different electrophoretic movement speeds in the same electric field. Generally, the more the net charge is, the smaller the particle is, and the closer it is to the sphere, the faster it moves in the electric field, and vice versa. In addition, the electrophoresis speed is also affected by the electric field strength, the pH of the solution, the ionic strength of the solution, the temperature, and the electrophoresis support.

Electrophoretic classification; There are many types of electrophoresis, but the basic principles are the same. Different electrophoresis has its own characteristics due to different supports or gels. According to the separation principle, it is divided into zone electrophoresis, shifting electrophoresis, isokinetic electrophoresis and gel electrophoresis. According to the presence or absence of solid support, it is divided into free electrophoresis and support electrophoresis. According to the form of the electrophoresis tank, it is divided into vertical electrophoresis types such as vertical, horizontal, columnar, and capillary.

Gel electrophoresis Gel electrophoresis using agarose and polyacrylamide gels [5,6] as the medium is most common in all electrophoretic media.

Agarose is a chain polysaccharide extracted from agar and combined with galactose and 3.6-anhydrogalactose. Agarose gel has a large pore size and is mainly used for electrophoretic separation of large molecules such as nucleic acids or proteins. And analysis, has the following advantages: (1) The amount of liquid is large, up to 98-99%, which is approximately free electrophoresis, but the diffusion degree of the sample is smaller than that of free electrophoresis, and the adsorption of DNA, RNA and protein is extremely small. (2) Agarose has the advantages of uniform support, neat zone, high resolution and good repeatability. (3) The electrophoresis speed is fast. (4) Good light transmission, can be directly measured by UV or other light source. (5) The zone is easy to dye and is advantageous for preparation.

The polyacrylamide gel is a macromolecule formed by polymerizing acrylamide and hydrazine, hydrazone bis-acrylamide. Polyacrylamide gel has a relatively small pore size and is mainly used for electrophoretic separation and analysis of proteins and small molecular substances. It has the following advantages: (1) Polyacrylamide gel has no or few side groups with ions, so electricity The osmotic effect is small and it is difficult to interact with the sample. (2) Since polyacrylamide gel is a synthetic substance, the concentration ratio of the monomer can be adjusted before polymerization to form different degrees of interchain structure, and the void fraction can be varied within a wide range, and can be To separate the size of the material molecules, choose the appropriate gel composition. In general, a gel containing 7-7.5% of acrylamide is suitable for the separation of molecular weights ranging from 10,000 to 1 million. For 10,000 or less, gels containing 15-30% of acrylamide are used. (3) Polyacrylamide is stable to heat in a certain concentration range. (4) The gel is colorless, transparent, and has excellent light transmission. It is easy to observe and can be directly measured by a detector.

Application of gel electrophoresis: It has been widely used for the analysis of various charged substances. In the experiment, the researchers can effectively separate the analyzed substances by the difference in molecular weight and the charge carried by electrophoresis. By comparison with molecular weight standards, the molecular weights analyzed can be accurately estimated and multiple samples can be analyzed simultaneously, isolated and purified.

Comparison of existing gel electrophoresis devices and techniques Gel electrophoresis is often used for the analysis of biological macromolecules such as proteins, DNA and RNA. Gel electrophoresis analysis of conventional DNA or RNA [6] is shown in Figure 1. Since DNA or RNA itself cannot fluoresce, it is necessary to prepare an agarose gel plate and add a DNA or RNA-binding fluorescent agent, ethidium bromide. Place the prepared gel plate in an electrophoresis tank, add DNA or RNA samples, and perform electrophoresis. DNA or RNA binds to ethidium bromide during electrophoresis, but the stained sample does not appear under visible light. It is necessary to exhibit a yellow-green fluorescent band spectrum after being excited by ultraviolet light (incident light) in a dark room. The fluorescence band spectrum can be filtered by an appropriate filter and then imaged with an optical or digital camera. Although ethidium bromide has long been known to be a strong carcinogen and UV is harmful to humans, there are no other better alternatives. Searching the Internet search engine for the term "gel electrophoresis", you can find many domestic and foreign manufacturers that produce and sell electrophoresis tanks, electrophoresis devices, UV light boxes and imaging or imaging systems. However, almost all devices and instruments are designed and manufactured in accordance with this traditional method of operation.

U.S. Patent No. 6,512,236 discloses a technique for visualizing fluorescent materials using visible blue light [7], and a commercial light source made by this technique is called Dark Reader [8]. A similar device produced by a domestic manufacturer is called the Blue Shield series. Visible gel transilluminator [9]. This technique uses a blue filter to filter the variegated light of an ordinary fluorescent lamp to obtain blue light. The blue light activates a fluorescent substance that uses blue light as the incident light, and the fluorescent substance is filtered by another filter plate to be observed by the naked eye or recorded by the camera. This device replaces the traditional UV light box for DNA or RNA electrophoresis analysis to avoid the use of ethidium bromide and ultraviolet light harmful to humans. However, this patent mainly deals with the production of blue visible light boxes. The ribbon is invisible during electrophoresis and needs to be transferred to a blue light box for observation before deciding whether to continue electrophoresis or photographic recording. The device must use blue and yellow-brown filter plates to observe and record fluorescent substances that require blue light as incident light, but cannot display fluorescent materials that excite (incident) light other than blue light. .

Gel electrophoresis analysis of proteins is usually performed using a vertical electrophoresis analysis device (Fig. 2A). When it is desired to examine fluorescent proteins (the proteins themselves fluoresce) or fluorescently labeled proteins or other probes, the vertical electrophoresis analysis device itself is not capable of visualizing these fluorescent materials, and other expensive detection devices are often required (Fig. 2B). Therefore, there is a need for a novel electrophoretic analysis device for analyzing fluorescent proteins or fluorescent-based immunoassays or molecular biological analyses. Summary of the invention

It is an object of the present invention to provide a multi-purpose gel electrophoresis analysis apparatus capable of real-time, continuous monitoring, comprising: a light transmission analysis plate and a narrow spectrum or pure spectral light source placed on both sides of the light transmission analysis plate, wherein The light emitted by the light source is incident at a horizontal angle to the gel plate.

In a preferred embodiment, the apparatus further includes an electrophoresis power source, an electrophoresis analysis tank.

In a preferred embodiment, a semiconductor light emitting diode is used as the light source to produce light of higher spectral purity. In another preferred embodiment, a laser tube is used to create a narrow spectrum or pure spectral source. Instead of a conventional light source, a narrow spectrum or pure spectral source can be used without a filter. This design allows the volume of the excitation source of the phosphor to be greatly reduced, allowing it to be integrated into the analytical system. A narrow spectrum or pure spectral source is a source that can be transformed. The choice of source wavelength can be based on the actual fluorescent dye used.

In a preferred embodiment, the light transmissive analysis panel is made of a light transmissive material which may be selected from natural or synthetic light transmissive materials, gels or polymers or clear liquids.

In a preferred embodiment, the electrophoresis analysis tank is selected from the group consisting of a horizontal analysis electrophoresis tank and a vertical analysis electrophoresis tank. In a preferred embodiment, the light source is mounted on either side of the gel plate and the light is incident in a direction parallel to the gel plate to activate the phosphor therein. This design facilitates the uniform distribution of light on the gel plate, avoiding direct light to the human eye and the camera, improving detection sensitivity and making the activated fluorescence visible to the naked eye.

In another preferred embodiment, the gel electrophoresis analysis device of the present invention further comprises a camera system. Electrophoresis After the end of 06 000715, you can take photos of the electrophoresis results.

In still another aspect, the present invention provides a method for real-time, continuous monitoring of gel electrophoresis, which comprises using the above-described gel electrophoresis analysis device provided by the present invention, using a pure spectrum matched with a fluorescent dye added, to gel The plate is angled at an angle that activates the fluorescent dye, allowing the sample to visualize its position in the gel plate for real-time, continuous monitoring.

The method of the present invention also includes photographing the gel plate that satisfies the requirements as appropriate. The imager for photographing can be combined with the gel electrophoresis analysis device of the present invention into a system such as the above gel electrophoresis analysis system of the present invention. The imager can also be a separate part, depending on the situation.

In a preferred embodiment, the fluorescent material is selected from the group consisting of a natural fluorescent substance, a substance labeled with a fluorescent material, or combined with a fluorescent material.

Electrophoretic analysis of DNA, RNA or protein using the apparatus and method of the present invention has a number of advantages, including: (1) Since the device has its own light source, no darkroom is required. It can continuously observe DNA, RNA or protein electrophoresis bands in real time during electrophoresis. (2) Since the light source can be changed, the system can be paired with many new DNA and RNA fluorescent dyes to visualize DNA or RNA in electrophoresis. For example, SYBR Green 1 [10] manufactured by Molecular Probes, USA can avoid strong carcinogenesis. Ethyl bromide.

(3) The sensitivity of using new DNA and RNA fluorescent dyes is 5-10 times higher than that of ethidium bromide, reaching a level of 10 nanograms (l'Ong). (4) Using the visible light spectrum as the excitation light, the harmful effects of ultraviolet light on the human body and the sample can be eliminated. (5) Compared with the traditional electrophoresis analysis method, it is easy to operate and low in cost.

The new device is significantly superior to existing DNA and RNA gel electrophoresis analyzers in all respects, and Table 1 lists more comparisons.

Table 1. Comparison of traditional electrophoresis device 6] with convertible light source electrophoresis device

The electrophoresis analysis apparatus according to the present invention is also well suited for the following analysis; (1) Analysis of various fluorescent proteins (GFP) and derivatives thereof. (2) Determination of the reaction between the fluorescently labeled antigen and the antibody. (3) Determination of gene hybridization reaction based on fluorescent labeling. (4) and determination of interaction between DNA/RNA and protein.

Furthermore, since the light source of the present invention can be easily replaced with a light source of other wavelengths, it can be used for electrophoretic separation and purification of other substances not listed. DRAWINGS

Figure 1 shows a schematic diagram of commonly used DNA and RNA gel electrophoresis analysis. In Fig. 1, 1 denotes an electrophoresis power supply, 2 denotes an electrophoresis tank, 3 denotes an ultraviolet light box, 4 denotes an ultraviolet light tube, 5 denotes a digital camera or an optical camera, 6 denotes a gel plate, and 7 denotes a filter. A indicates that electrophoresis and dyeing were performed, but the ribbon was not visible. B indicates that the gel plate is placed in an ultraviolet light box and observed in a dark room to determine whether further electrophoresis or photographic recording is performed. C indicates that a photographic recording is performed.

Figure 2 shows a schematic diagram of the analysis of commonly used fluorescent proteins or proteins bound to fluorescent probes. In the figure, A denotes a vertical electrophoresis apparatus, and B denotes a bioluminescence analyzer or a fluorescence scanner. In A, 1 is electrophoresis Source, 2 is a fluorescent protein sample, 3 is an electrophoresis upper tank, 4 is a gel plate, 5 is an electrophoresis trough, 6 is an electrophoresis liquid, 7 is a protein transfer, 8 is a direct analysis of a fluorescent protein sample, and 9 is a Fluorescent probes for specific proteins were probed and analyzed by instrument.

Fig. 3 shows a specific gel electrophoresis analysis device integrated with a semiconductor light source and a light source which can be converted. The dotted line frame 1 indicates the instrument case, 2 indicates the digital camera, 3 indicates the optical plastic cover, and 4 indicates the multi-purpose electrophoresis tank that can change the light source. When the device is used for the test, the chromosomal DNA band on the gel can be directly observed with the naked eye, and the observation can be continuously performed, the incident light source can be changed, and the in-situ photographic recording can be performed.

Fig. 4 shows a schematic view of the components and structure of an electrophoresis device with a light source. A indicates a device that has been assembled and is undergoing electrophoresis. B shows the structure of the inside of the device with the gel plate after the cover with the filter is uncovered. C shows the structure in which the gel plate has been taken out but the light source is still retained. D shows the electric swimming pool after the gel plate and the light source plate are taken out. E is an electrophoresis power supply.

Fig. 5 is a view showing the relationship between the light source and the gel plate in the horizontal gel electrophoresis apparatus and the incident angle, wherein 1 represents a light source, 2 represents a gel plate, and an arrow with a double line indicates an incident direction of incident light.

Figure 6 shows a schematic view of the structure of a vertical gel electrophoresis apparatus with a switchable light source. 1 is the light source, 2 is the gel plate, 3 is electrophoresis, 4 is the electrophoresis upper tank, and 5 is the electrophoresis buffer. An arrow with a double line indicates the direction of incidence of the incident light source.

Figure 7 shows the application of blue visible light produced in U.S. Patent No. 512,236 to DNA analysis. The figure shows that firstly, A conventional method electrophoresis is required, then dyeing, B transfers the dyed gel plate to the blue light visible light box involved in the technology, and the sample can be observed and analyzed in the dark room to determine Continue electrophoresis or perform C photo recording. In the figure, 1 indicates gel plate dyeing, 2 indicates a fluorescent tube, 3 indicates a blue visible light box, 4 indicates a filter, 5 indicates a camera, and 6 indicates a gel plate.

Fig. 8 is a graph showing the results of experiments conducted by the present invention using a gel electrophoresis apparatus with a switchable light source for DNA analysis. In this experiment, DNA samples from 8 different sources were treated with nuclease (A) or without enzyme (B). The electrophoresis time was 45 minutes, and the experimental results were recorded with a digital camera.

Fig. 9 is a view showing an electrophoresis apparatus with a light source for separating and purifying a DNA fragment. A is a plan view of the gel plate aligned with the purification plate. 1 is the DNA band after electrophoresis separation, represented by a gray rectangle, 2 and 3 respectively represent the gel plate and the separation plate, and the dotted line indicated by a indicates the horizontal center line. B is a cross-sectional view of the horizontal midline of the gel plate and the purification plate, indicating the purification process of the DNA band. Among them, 1 indicates the case of assembly alignment; the DNA band to be recovered needs to be aligned with the groove in the separation plate, and the lower opening of the purification hole is sealed with a semi-permeable membrane 5 to prevent DNA from passing. 2 indicates the electroelution process, 3 indicates that the DNA stops when it moves to the purification well PT/CN2006/000715 Electrotransport, 4 indicates recovery of purified DNA samples. Since the electrophoresis device has a light source, the recovery of the sample can be observed in real time. detailed description

Electrophoresis tank The horizontal electrophoresis tank and the components for placing the light source can be fabricated as shown in Figures 3 to 4. The electrophoresis tank can be made of a transparent or opaque material, but the insulation is required to not leak. The vertical electrophoresis analysis tank can be used to create a vertical analysis electrophoresis tank as shown in Fig. 2.

The light source insert is made of a plastic mold as shown in Fig. 5. The light source inserts should be placed on both sides of the transparent analysis board. The side of the component on which the light source is placed facing the analysis plate must be transparent.

The light transmission analysis board, the analysis board is located between the light sources. The assay plate can be a solid, semi-solid or liquid material of a natural light transmissive material such as a gel made of agar or a artificially polymerized light transmissive material.

Light source: A replaceable narrow spectrum or pure spectral source is located in the light source insert. The emitted light must be incident at an angle parallel to the analytical (gel) plate. The choice of source wavelength depends on the fluorescent dye or fluorescent label chosen. Many manufacturers now produce fluorescent dyes or fluorescent labels. The pairing of fluorescent dyes with which wavelength source can be found in the product literature provided by the manufacturer [10] or in the commodity reference manual [3]. Those skilled in the art will be familiar with the knowledge of which fluorescent dyes are paired with which wavelength source. Table 2 summarizes the spectra of incident and emitted light of some commonly used fluorescent proteins and fluorescent compounds.

The fabrication of this device requires the use of narrow-spectrum or pure-spectrum light sources. With the development of semiconductor technology and optical technology, it has been easy to obtain a purer spectral light source through light-emitting diode (LED) or laser technology. Light-emitting diode technology is now able to produce all spectra from ultraviolet to infrared [11]. It is thus easy to obtain LEDs of various wavelengths. However, the LED or laser tube has a small light-emitting area, and it is necessary to fabricate and test LEDs of a specific shape, illumination angle and brightness, and assemble them in a certain distance in order to obtain uniform incident light.

The incident and emitted light of commonly used fluorescent compounds and fluorescent proteins 3 , 10

Color filter (plate). With the special design of the present invention, it is completely unnecessary to use a color filter or a color filter to obtain a specific incident light source, and fluorescence can be observed with the naked eye and photographed. However, use a color filter or color filter that only allows fluorescence (emitted light) to pass between the fluorescent sample and the observer or camera. It may be possible to improve sensitivity. The color filter may be a flat cover, or may be in the form of glasses, or may be placed on the front lens of the camera. The color and filter characteristics of the color filter depend on the wavelength of the emitted light of the fluorescent dye or fluorescent label selected. The principle of selection is to block the passage of incident light but pass the fluorescence as much as possible.

The present invention is not only applicable to electrophoretic analysis of nucleic acids, but can be fabricated using the principles mentioned in the present invention as long as it is based on fluorescent labeling and fluorescence reaction. Here, it is mentioned that the fluorescent label and the fluorescent reaction include natural fluorescent substances such as various fluorescent proteins and various derivatives, and artificially synthesized fluorescent compounds; such as fluorescein.

An example of a horizontal gel electrophoresis analysis apparatus fabricated by the present invention is shown in Fig. 3. The dotted line in the figure indicates the instrument case, and the upper part inside the case is a digital camera. It is of course also possible to replace the digital camera with an optical camera. Below the digital camera is an optical plastic cover with a gel electrophoresis analysis device with a switchable light source.

3 to 5 show an example of the horizontal gel electrophoresis analysis apparatus of the present invention and its working principle. Figure 5, where E is the electrophoresis power source, A is the gel electrophoresis device with the light source, B is the cover that can be removed with the color filter, C is the electrophoresis tank with the gel removed, and D is the electrophoresis from which the switchable light source has been removed. groove. In other words, in the specific operation, the electrophoresis tank may first exist in the state indicated by D. The technician can install the pure spectrum source and then put the gel plate; or you can make the glue first and then the light source. Then add the electrophoresis solution and add the sample. The cover with the color filter is then covered, and finally the power is applied for electrophoresis.

The vertical gel electrophoresis analysis device works similarly to a vertical gel electrophoresis analysis device. Figure 6 shows an assembly schematic of a vertical gel electrophoresis apparatus with a switchable light source and the direction of incidence of the light source.

During electrophoresis, light from a purely spectral source is incident on the plate parallel to the gel plate, and the gel plate is used as a light guiding medium to evenly distribute the light in the gel plate. The incident light activates the phosphor in the gel plate to visualize the position of the sample in the gel plate. Thus, the technician can monitor the progress of electrophoresis in real time and continuously. Stop electrophoresis when appropriate. The gel plate can be photographed as needed.

The apparatus of the present invention and its method of use will be described in detail below. It should be understood that these specific embodiments are merely illustrative and are not to be considered as limiting. Example 1 : Agarose gel electrophoresis analysis for DNA

DNA sample analysis was performed using the non-carcinogenic fluorescent dye SYBR Green 1 [10] sold by Molecular Probes, USA. The procedure is as follows;

1. The light source of the system uses a 490 nm LED light source.

2. Prepare an agarose gel plate without any DNA stain according to the molecular weight of the analyzed molecules (the traditional method requires the insertion of carcinogenic ethidium bromide as a DNA stain in the gel plate or buffer). The relationship between the concentration of the selected agarose gel plate and the molecular weight of the DNA to be analyzed can be found in related reference books [12, 13] and is well known to those skilled in the biological arts.

3. Mix the appropriate amount of DNA with the loading solution with SYBR Green I fluorescent stain.

4. Add the running buffer to the electrophoresis tank.

5. Add the mixture from step 3 to the sample well of the gel plate.

6. Connect the electrophoresis tank to the electrophoresis unit. The loading end is the cathode end. The electrophoresis was performed at a length of 8-10 volts/cm.

7. During electrophoresis, the progress of electrophoresis can be observed in real time by using the color filter in the middle of the cover or by electrophoresis.

8. Record the results of the sample when it is electrophoresed to the desired distance. Figure 8 shows the results of a typical DNA gel electrophoresis analysis. Example 2: Electrophoretic separation and purification for DNA (schematic diagram is shown in Figure 9)

Every laboratory engaged in gene cloning and gene expression separates purified DNA fragments from electrophoretic gel plates. Separation and purification of DNA fragments from gels generally requires the purchase of a kit of reagents and is inefficiently purified. Since the electrophoresis apparatus produced by the present invention can observe the progress of electrophoresis in real time by the naked eye or through the color filter of the electrophoresis cover. Therefore, purification of DNA samples is very simple and reliable. The procedure for separating and purifying DNA samples using a 490 nm purification unit is as follows:

1. The light source of the system uses a 490 nm LED light source.

2. Digest DNA samples with appropriate restriction enzymes according to the general principles of molecular biology [12,

13].

3. Make agarose gel plates without any stain according to general principles in the field of molecular biology.

4. Add 1/10 of the sample solution to the digested sample and mix. The sample solution contained DNA fluorescent staining SRBR Green I.

5. Add the mixed sample to the sample well of the gel plate.

6. Add the electrophoresis buffer to the electrophoresis tank and connect to the electrophoresis apparatus. The loading end is the cathode end. Electrophoresis was performed at a length of 8-10 volts/cm.

7. Observe the progress of the electrophoresis in real time with the naked eye or through the filter plate of the electrophoresis cover.

8. When the desired DNA separation is achieved, stop the electrophoresis and transfer the gel plate to the separation plate. The DNA band that needs to be recovered is moved and aligned with the groove of the purification plate. 9. Connect the upper side of the purification plate to the negative electrode and the lower side to the anode electrode. Electrophoresis was carried out by running a gel length of 4-6 volts/cm. The progress of electrophoretic separation can be observed in real time with the naked eye or through a color filter.

10. Turn off the power supply after fully electrophoresis with the fluorescent DNA band into the separation hole. Remove the gel plate and pipette the sample from the separation well to the small tube.

11. Directly add the enzyme buffer to the DNA fragment and the ligase, or concentrate the sample with ethanol and connect with ligase. The technique for ethanol precipitation of DNA can be found in related reference books [12, 13] and is well known to those skilled in the biological arts.

12. Subsequent operations are performed according to standard procedures for molecular cloning [12, 14]. Example 3: Electrophoretic analysis for green fluorescent protein 405

Green fluorescent protein has been widely used as a reporter gene in modern biotechnology. Expression of green fluorescent protein is usually determined by fluorescence microscopy analysis. The detection of traditional green fluorescent protein requires an 405 nm incident light source. Since the incident light source is not disposed in a general fluorescence microscope, the presence of this type of fluorescent protein cannot be observed. However, the electrophoresis apparatus fabricated by the present invention is easily observed. The specific operation steps for analyzing and detecting the conventional green fluorescent protein 405 using an electrophoretic analysis system with a switchable light source are as follows;

1. Convert the system's incident light source to a light source with a wavelength of 405 nanometers.

2. Make non-denaturing cell melts according to standard procedures in cell biology.

3. Make 1% agarose gel plate or 10% polyacrylamide gel plate without any stain.

4. Add the appropriate amount of cell lysate (typically 10-25 μg) to the sample well of the gel plate.

5. Add the electrophoresis buffer to the electrophoresis tank and connect to the electrophoresis apparatus. The loading end is the cathode end. Electrophoresis was performed at a length of 8-10 volts/cm.

6. During the electrophoresis process, the progress of electrophoresis can be observed in real time with the naked eye or through a color filter.

When the sample is electrophoresed to the desired distance, the experimental results are recorded using an optical or digital camera. The experimental implementation process shows that the electrophoresis can be observed continuously and in real time by using the apparatus and method of the present invention, and the operation is simple. Example 4: Analysis of DNA-protein binding reaction

Since the interaction between DNA and protein or RNA and protein plays a very important role in regulating cell growth, differentiation and cell function, the binding reaction of DNA/protein or RNA/protein is commonly used in vitro as a hot spot in biological research. One [15-17]. The principle of measurement is that when a specific DNA or RNA fragment binds to a protein, its mobility in the polyacrylamide gel will be less than that of the unbound DNA fragment, thereby detecting an activated DNA-binding protein regulatory factor. Common measurement techniques need to be put The radioisotope visualizes the presence of a protein-bound probe, or biotin or a digoxin-labeled DNA probe instead of an isotope-labeled probe that binds to the protein. However, the use of non-isotopic methods requires some subsequent steps to be able to visualize the position of the probe bound to the protein. These subsequent visualization steps are time consuming and expensive. Using the vertical electrophoresis apparatus (shown in Figure 6) of the present invention in combination with the fluorescent material Cy5-labeled probe, the DNA/protein binding reaction can be monitored and measured in real time in the gel electrophoresis analysis process, thereby The measurement is greatly simplified and the cost is saved. The specific operation steps are as follows;

1. Prepare or pass the DNA probe labeled with the fluorescent compound Cy5, which is commercially synthesized by Biotech.

2. Replace the incident light source used in the system with a wavelength of 650 nm.

3. Prepare 1.4-1.8% agarose gel plates or 5-6% polyacrylamide gel plates without any stain according to general principles in the field of molecular biology [12,16].

4. Combine the cell extract with the fluorescent probe in a test tube according to the product manual or the standard method described in the textbook [16].

5. Add 1/10 of the DNA sample commonly used in the laboratory to the reaction tube and mix.

6. Add the mixed sample to the sample well of the gel plate.

7. Add the electrophoresis buffer to the electrophoresis tank and connect to the electrophoresis apparatus. The loading end is the cathode end. Electrophoresis was carried out at a gel length of 8-10 volts/cm.

8. Observe the progress of the electrophoresis in real time by the naked eye or through the color filter of the electrophoresis cover.

9. Record the results of the experiment with an optical or digital camera when the sample is electrophoresed to the desired distance.

The experimental implementation shows that the electrophoresis can be observed continuously and in real time by the apparatus and method of the present invention, and the operation is simple. Reference material

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2. Joseph R. Lakowicz, Richard B. Thompson (Editor, Advances in Fluorescence Sensing Technology V "Progress in Biomedical Optics and Imaging, SPIE-International Society for Optical Engine 2001."

3. Mary- Ann Mycek, Brian W. Pogue, Handbook of Biomedical Fluorescence, Marcel Dekker 2003. 4. Hassan M, Klaunberg BA, "Biomedical applications of fluorescence imaging in vivo." Comp Med. 2004, 54: 635-44.

5. B. David Hames, B. D. Hames, D. Rickwood, "Gerro Electrophoresis of Proteins: A Practical Approach (Practical Approach Series)) Oxford University Press; 3rd edition 1998.

6. Robin Martin, "Gel Electrophoresis - Nucleic Acids: Nucleic Acids (Introduction to Biotechniques), BIOS Scientific Publishers 1996.

7. "Fluorometric detection using visible light", U.S. Patent No. 6,512,236.

8. See website http://www.clarechemical.com/transilluminator.htm o

9. See website http://www.shinegene.org.cn/2004/ld 001.html.

10. "Molecular Probes, The Handbook - A Guide to Fluorescent Probes and Labeling Technologies", 9th edition, Invitrogen 2003.

1 1. E. Fred Schubert, "Light-Emitting Diodes", Cambridge University Press 2003.

12. Lu Shengdong Modern Molecular Biology Experimental Technology Beijing: China Union Medical University Press, 1999.

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Claims

Claim

What is claimed is: 1. An electrophoretic analysis device, comprising: a light transmitting analysis plate; and a narrow spectrum or pure spectral light source disposed on both sides of the light transmitting analysis plate, wherein the light emitted by the light source is at a level with the gel plate The angle of the shot.

2. The electrophoretic analysis apparatus according to claim 1, wherein a narrow spectrum or pure spectrum light source is generated using a light emitting diode or a laser tube.

The electrophoresis analysis apparatus according to claim 1, wherein the narrow spectrum or pure spectrum light source is a light source that can be converted.

The electrophoresis analysis apparatus according to claim 1, wherein the light transmission analysis plate is made of a light transmissive material.

5. The electrophoresis analysis device according to claim 4, wherein the light transmissive material is selected from a natural or synthetic light transmissive material, a gel or a polymer, or a transparent liquid.

6. The electrophoresis analysis device according to claim 1, wherein the device further comprises a photographic device and a filter.

The electrophoresis analysis apparatus according to claim 1, wherein the electrophoresis analysis tank is selected from the group consisting of a horizontal analysis electrophoresis tank and a vertical analysis electrophoresis tank.

8. A method for real-time, continuous monitoring of gel electrophoresis, characterized in that the method comprises performing electrophoresis using the electrophoresis analysis device according to claim 1, using a pure spectrum matched with the added fluorescent dye to condense The horizontal angle of the rubber sheet is injected, which activates the fluorescent dye, allowing the sample to visualize its position in the gel plate for real-time, continuous monitoring.

9. A method according to claim 8 wherein the fluorescent material is selected from the group consisting of a natural fluorescent substance, a substance labeled with a fluorescent material or combined with a fluorescent material.

10. The method of claim 8 further comprising taking a picture of the resulting electrophoresis result.