Electrophoretic apparatus

Abstract: An electophoretic apparatus for determining the base sequence of DNA has a device for introducing a sample, a device for controlling the temperature of the sample in the sample introducing device, a device for causing electrophoresis of the temperature-controlled sample in a gel to separate it into fragments, and a device for analyzing those fragments. The apparatus makes it possible to carry out successively and automatically a process which determines the base sequence of DNA by treating DNA in accordance with the Sanger method to form complementary DNA fragments of different lengths ending specifically with four different kinds of bases, separating those fragments from one another electrophoretically, and identifying the base at the end of each of those fragments.

Description: BACKGROUND OF THE INVENTION This invention relates to a gel electrophoresis apparatus. There is known an arrangement for an electrophoretic process which employs a slab or capillary gel for determining the sequence of bases in DNA. The sequence of bases in DNA is determined by the Sanger method which employs an M13 vector, or plasmid PUC. The Sanger method which employs plasmid PUC, however, often makes it difficult to decode the sequence of bases in DNA correctly because of the stacking of DNA's modified by plasmid PUC and having a single strand. There has, therefore, been proposed an arrangement for a process which employs the Sanger method with the PCR method which employs polymerase having a high temperature. DNA is thermally modified into single-strand, or template DNA, and a primer is annealed onto the template DNA. Then, the Sanger method is applied to the template DNA to synthesize thereon a complementary DNA fragment of any length ending with four different kinds of bases. The PCR method is applied to the template DNA on which the complementary DNA fragment has been formed, so that the template DNA may be separated from the complementary DNA fragment. The annealing of the primer onto the template DNA, and the Sanger and PCR methods are repeated several tens of times to produce a large amount of complementary DNA fragments, whereby an improved accuracy of electrophoretic identification can be achieved to determine the sequence of bases in DNA correctly. In the process which employs the Sanger method in combination with the PCR method, however, the electrophoretic operation is completely separated from the treatment of a sample by the Sanger method. The sample is treated by the Sanger method, and is transferred by a pipette, or the like to a electrophoretic apparatus. As there is no alternative but to do any such work manually, the above process cannot automatically determine the sequence of bases in DNA. In F. Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467 a method for determining nucleotide sequences in DNA is described, which makes use of the 2', 3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. F. Toneguzzo at al., BioTechniques 7 (1989) 866-877 describe a system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis according to the Sanger chain termination method. During the electrophoresis 32 P-labeled DNA fragments are detected by solid state detectors. US-A-5074981 describes a method and apparatus for high speed gel eletrophoresis employing elevated voltage values and relatively short time periods by using a gel block having special geometry and including sample wells of triangular cross-section, whereby the temperature throughout the gel is continuously monitored at various points in the gel; the heat, generated by employing elevated voltage values is withdrawn from the described gel block in a specific manner. SUMMARY OF THE INVENTION It is, therefore, an object of this invention to enable the treatment of a sample at a controlled temperature and the electrophoretic treatment thereof to be carried out successively and automatically. It is another object of this invention to enable the base sequence of DNA to be determined automatically. According by there is provided an electrophoretic apparatus according to claim 1; preferred embodiments of the inventive apparatus are covered by claims 2 to 5. The apparatus makes it possible to carry out automatically a series of operations including the treatment of the sample at a controlled temperature and the electrophoretic treatment thereof. According to this invention, there is provided an apparatus for determining the base sequence of DNA with which the following steps can be performed: the steps of treating DNA in accordance with the Sanger method to form complementary DNA fragments of different lengths ending specifically with four different kinds of bases; separating the fragments from one anther by electrophoresis; and identifying the base at the end of each of the fragments, the step being carried out successively and automatically. It is easier to determine the base sequence of DNA by the apparatus of this invention than by the conventional arrangement in which the step of forming complementary DNA fragments and the step of separating them by electrophoresis are carried out independently of each other. The apparatus enables the automatic sequencing of bases in DNA by carrying out the treatment of the sample at a controlled temperature and the electrophoretic treatment thereof successively. These and other objects and advantages of this invention will become more apparent from the following description and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagrammatic representation of an electrophoretic apparatus embodying this invention; and Fig. 2 is a graph showing a temperature cycle employed in the apparatus. DESCRIPTION OF THE PREFERRED EMBODIMENT An electrophoretic apparatus embodying this invention is shown in Fig. 1. The apparatus is intended for use in determining the base sequence of DNA in accordance with the Sanger method. The apparatus consists mainly of a sample introducing device 2 and an electrophoretic vessel 3 provided at the top and bottom, respectively, of a glass capillary 1. The capillary 1 holds a polyacrylamide gel. The sample introducing device 2 is adapted for receiving the supply of a sample of DNA, DNA polymerase and a Sanger reagent, as well as an electrolytic buffer solution 4. The electrophoretic vessel 3 is adapted for holding the buffer solution 4. The sample introducing device 2 and the electrophoretic vessel 3 are each provided with an electrode 6 which is connected to a high-voltage power-supply device 7. A heating and cooling device 8 is connected to the sample introducing device 2 for heating or cooling the mixture of the sample and the reagent therein. The sample introducing device 2 is provided with a temperature sensor 9. The heating and cooling device 8 and the temperature sensor 9 are connected to a temperature controller 10 which controls the heating and cooling device 8 in accordance with the output of the temperature sensor 9. A device 11 for supplying the buffer solution has a supply pipe 11a terminating in the sample introducing device 2. A device 12 for controlling the supply of the buffer solution is connected to the buffer solution supplying device 11 for controlling it so that it may supply the buffer solution into the sample introducing device 2 upon completion of the treatment of the sample therein. A fluorescent detecting device 13 is positioned across the middle portion of the glass capillary 1 for detecting the fluorescent pigment combined with the end of each of DNA fragments separated by electrophoresis in the capillary 1. The fluorescent detecting device 13 consists mainly of a laser 14, a converging lens 15 for converging in the capillary 1 light emitted by the laser 14, a lens 16 for diverging into parallel rays the light leaving the capillary 1, an interference filter 17 for detecting the wavelength of the light leaving the lens 16, and a photomultiplier (PM) 18 for detecting the light leaving the filter 17. The temperature controller 10, the device 12 for controlling the supply of the buffer solution, the high-voltage power-supply device 7, the laser 14 and the photo-multiplier 18 are connected to a data control analyzer 19 which controls them and determines the base sequence of DNA in accordance with the output of the photomultiplier 18. Description will now be made of a process in which the electrophoretic apparatus as hereinabove described is used to determine the base sequence of DNA. The sample introducing device 2 is supplied with a mixed solution containing DNA of which the base sequence is to be determined, a primer with which a fluorescent substance, such as FITC, is combined, a Sanger reagent, and DNA polymerase required for the PCR reaction. The heating and cooling device 8 is controlled by the temperature controller 10 to control the temperature of the mixed solution in the sample introducing device 2. More specifically, the solution is maintained at a temperature of 95°C for about one minute, so that the thermal modification of plasmids may cause the conversion of DNA to single-strand, or template DNA. The solution is cooled to a temperature of 55°C, and maintained at that temperature for about 30 seconds, so that the primer may be annealed onto the template DNA. Then, the solution is heated again to a temperature of about 75°C, and maintained at that temperature for about one to three minutes, so that complementary DNA fragments of different lengths ending specifically with four different kinds of bases may be formed on the primer on the template DNA by the Sanger reaction. The temperature cycle as hereinabove described is repeated. When the solution is heated to 95°C, and maintained at that temperature for about one minute, the template DNA and the complementary DNA fragments are separated from each other. When the solution is cooled to 55°C, the primer is annealed onto the template DNA again. And when the solution is heated to about 75°C again, the Sanger reaction takes place again to form complementary DNA fragments on the template DNA. The cycle is repeated, for example, 10 to 30 times until the solution is finally maintained at 70°C for seven minutes, whereby a large amount of complementary DNA fragments of different lengths are produced. Upon completion of the repeated cycle, the buffer solution 4 is supplied from the buffer solution supplying device 11 to the sample introducing device 2, and a high voltage is applied across the electrodes 6 by the high-voltage power-supply device 7. The complementary DNA fragments of different lengths in the sample introducing device 2 start electrophoretic movement through the glass capillary 1, and are separated from one another in the polyacrylamide gel, beginning with ones of short length, and resulting in the formation of a DNA band 20, as shown in Fig. 1. Each DNA band 20 emits fluorescent light upon application of laser light, as the complementary DNA fragments forming it contain a fluorescent pigment. Wherein the wavelength of the fluorescent light emitted by each DNA band 20 is determined by the photomultiplier 18, it is possible to identify the base at the end of the complementary DNA fragment forming the DNA band 20. Wherein the bases at the ends of the complementary DNA fragments are determined one after another from the lower DNA bands 20, it is possible to determine the base sequence of DNA. The electrophoretic apparatus as hereinabove described makes it possible to carry out successively the Sanger treatment of the sample combined with the PCR method and the electrophoretic treatment thereof, and thereby determine the base sequence of DNA automatically. Although the invention has been described with reference to the electrophoretic apparatus or process employing a capillary gel, it is equally applicable to an apparatus or process employing a slab, or other gel, too. Although the base sequence of DNA has been described as being determined by a fluorescent system, this invention is equally applicable to an apparatus or process employing another detecting system relying upon, for example, a mass spectrum, too.

Claims: A gel electrophoresis DNA sequencing apparatus for forming according to the Sanger method base-specific terminated DNA fragments complementary to DNA templates to be sequenced, and for simultaneously employing the PCR method for amplifying the base-specific terminated DNA fragments, the apparatus comprising: a sample introducing device (2) arranged to carry out polymerase chain and Sanger reactions on sequencing sample DNA, which may be a fluorescent dye-labeled DNA, said sample introducing device (2) for introducing products of the polymerase chain and Sanger reactions into a gel electrophoresis means (1); and means (9, 10) for sensing and controlling the temperature of said sample in said sample introducing device (2); the gel electrophoresis DNA sequencing apparatus characterized in further comprising: a gel electrophoresis means (1) for causing the electrophoresis of said temperature-controlled sample in a gel to seperate said sample into fragments having an end joined to said sample introducing device (2); and a data control analyzer (19) connected to said temperature sensing (9) and controlling means (10), and to said gel electrophoresis means (1), and means (14, 15, 16, 17, 18) for analyzing said fragments. A gel electrohoresis DNA sequencing apparatus as set forth in claim 1, further comprising a heating and cooling device (8) for heating or cooling the mixture of the sample and the reagent in the sample introducing device. A gel electrophoresis DNA sequencing apparatus as set forth in claim 2, wherein said temperature sensing (9) and controlling means (10) comprises: a temperature sensor (9) in said sample introducing device (2); and a temperature controller (10) for controlling said heating and cooling device (8) and said temperature sensor (9) being connected to said temperature controller (10). A gel electrophoresis DNA sequencing apparatus as set forth in claim 3, further comprising: means (11) for supplying a buffer solution to said sample introducing device (2); and a buffer supply controller (12) connected to said buffer solution supply device (11). A gel electrophoresis DNA sequencing apparatus as set forth in claim 4, wherein: the gel electrophoresis means comprises a gel electrophoresis glass capillary (1) having one end joined to said sample introducing device (2) and an opposite end in a buffer solution vessel (3), and having a pair of electrodes (6) in said sample introducing device (2) and said buffer solution vessel (3), the pair of electrodes (6) connected to a light-voltage power supply (7); and said data control analyzer (19) is arranged to direct, upon completion of the polymerase chain reaction and of thermal cycling of the Sanger reactions, said buffer supply controller (12) to supply buffer solution to the sample introducing device (2), and said high-voltage power supply (7) to apply a high-voltage across the electrodes (6) to initiate electrophoretic movement of the sequencing fragments in said sample introducing device (2) through said gel electrophoresis capillary (1).

Inventors: MOCHIZUKI TAKANORI, MOCHIZUKI, TAKANORI
Applicants: SHIMADZU CORP, SHIMADZU CORPORATION
Application year: 1993
Main classification: G01N27
IPC codes: C12Q1, G01N27, C12N15