JPH09281078A - DNA base sequencer - Google Patents

Abstract

(57) An object of the present invention is to provide a miniaturized DNA nucleotide sequencer using an inexpensive light source and an inexpensive light receiving system. A vertically-held flat plate gel electrophoretic means having a large number of fluorescently labeled DNA fragment migration paths, an excitation light source for irradiating the migration paths of the electrophoresis means with excitation light, and the excitation light In the DNA base sequence determination device, which comprises fluorescence detection means for detecting fluorescence generated from the fluorescence-labeled DNA fragment irradiated by and converting it into an electric signal, the excitation light source is a high-intensity light emitting diode (LED),
The fluorescence detecting means comprises a gradient index lens array, a filter, and a CCD line sensor, and the high-intensity light emitting diode is connected to a temperature control circuit having an electronic cooler. Decision device.

Description

Detailed Description of the Invention

[0001]

[0001] The present invention relates to a DNA base sequencer. More specifically, the present invention relates to an apparatus capable of efficiently and rapidly determining the base sequence of DNA using a fluorescent label.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining a base sequence of DNA or the like.

Conventionally, when performing electrophoresis, a sample is labeled with a radioisotope and analyzed, but this method has a drawback that it requires time and effort. In addition, maximum safety and control are always required from the viewpoint of radioactivity management, and analysis cannot be performed unless in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, a fluorescently labeled DN is used.
The A fragment is allowed to migrate in the gel.
A photoexciting section and a photodetector are provided for each electrophoresis path below ２０20 cm, and DNA fragments passing therethrough are sequentially measured. For example, DNAs of various lengths whose terminal base species are known are duplicated by an enzymatic reaction (dideoxy method) using a DNA chain whose sequence is to be determined as a template, and these are labeled with a fluorescent substance. That is, adenine (A) labeled with a fluorescent substance
A fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group are obtained. These fragment groups are mixed and injected into separate electrophoresis lane grooves of an electrophoresis gel,
Apply voltage. Since DNA is a chain polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Shorter (small molecular weight) DNA strands move faster and longer (larger molecular weight) DNA chains move more slowly, so that DNA can be fractionated by molecular weight.

Japanese Patent Application Laid-Open No. 63-21556 discloses that a line on a gel of an electrophoresis apparatus irradiated with a laser and an arrangement direction of a photodiode array are perpendicular to a migration direction of a DNA fragment in the electrophoresis apparatus. Is disclosed.

FIG. 14 is a schematic diagram for explaining the structure of the apparatus. In the apparatus shown in FIG. 14, the laser light emitted from the light source 70 is reflected by the mirror 72, and a certain point in the gel of the electrophoretic plate 74 is irradiated with the laser light horizontally from the side. When the fluorescently labeled DNA fragment 76 that has migrated in the gel passes through this irradiation region, fluorescence is sequentially emitted from this DNA fragment. At this time, the type of the terminal base can be determined from the horizontal position of the fluorescence emission, the length of the fragment can be determined from the difference in the migration time from the start of the migration, and the sample can be further identified by the emission wavelength. The fluorescent light from each migration path forms an image at the light receiving section 82 of the image intensifier 80 by the lens 78. This signal is amplified, converted into an electric signal by the photodiode array 84, and measured. The sequence of each DNA fragment is calculated by processing the measurement result by computer, and the base sequence of the target DNA is determined.

As personalization of DNA base sequence determination devices is progressing, cost reduction, size reduction and performance (analyzed base length) improvement of the device are becoming the most important issues. The apparatus shown in FIG. 14 uses an argon laser or a He—Ne (Y) laser as a light source, and uses a light receiving system image intensifier. Due to the relatively short life of laser light sources, they must be replaced regularly to maintain consistent performance. However, since the laser light source is expensive, replacement thereof requires a great expense. Further, the laser light source requires a power source for driving in addition to the oscillation device, which is a bottleneck in downsizing the device. Further, the image intensifier used for the light receiving system is not only expensive, but also a relatively large device, which has been an obstacle to downsizing of the DNA base sequence determination device.

[0008]

Therefore, an object of the present invention is to downsize a DN by using an inexpensive light source and an inexpensive light receiving system.
It is to provide an A base sequence determination device.

[0009]

[Means for Solving the Problems] The above-mentioned problems are solved by a plate-type gel electrophoretic means which is held vertically and has a large number of electrophoretic paths for fluorescently labeled DNA fragments, and excitation light in the electrophoretic paths of the electrophoretic means. In the DNA base sequence determination device, the excitation light source emits high-luminance light, and the fluorescence detection means detects fluorescence generated from the fluorescence-labeled DNA fragment irradiated by the excitation light and converts it into an electric signal. A diode (LED), the fluorescence detecting means includes a gradient index lens array, a filter, and a CCD line sensor, and the high-intensity light emitting diode is connected to a temperature control circuit having an electronic cooler. Is solved by a DNA nucleotide sequencer characterized by the following.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a partial schematic perspective view showing a schematic configuration of an example of a DNA nucleotide sequence determination device of the present invention. In the DNA nucleotide sequencer of the present invention, the electrophoresis plate 74
The high-intensity light emitting diode group 1 as the excitation light source and the fluorescence detecting means 2 are arranged so as to face each other with sandwiching therebetween. FIG. 2 is a plan view showing a positional relationship among the high-intensity light emitting diode group 1 of the excitation light source, the migration plate 74, and the fluorescence detecting means 2.

As shown in FIG. 2, a plurality of high-intensity light emitting diodes are arranged in a line to form an excitation light source 1. As described above, since the DNA is composed of adenine (A), cytosine (C), guanine (G) and thymine (T), these four types of bases form one DNA sample. Therefore, as shown in FIG.
Four migration paths 86 are used for each DNA sample. In the present invention, one per 1 DNA sample is
High brightness LEDs are used. That is, one high-intensity light emitting diode covers four migration paths.

In FIGS. 1 and 2, the fluorescence detecting means 2 is, for example, a gradient index lens array 3 and a fluorescence filtering means 5 in which two filters are stacked (described in detail below).
And a solid-state image sensor, for example, a CCD (charge coupled device) line sensor 7. When the excitation light from the high-intensity light emitting diode 1 is radiated from the front to the DNA fragments 76 that have migrated downward along the migration paths 88 of the gel electrolyte layer 86 of the migration plate 74, fluorescence is generated. This fluorescence enters the gradient index lens array 3, and the light that has passed through this lens array then enters the filter 5.
Excitation light, stray light, background light, or the like having a wavelength other than the fluorescence component is cut by this filter. afterwards,
The fluorescence that has passed through the filter is received by the CCD line sensor 7 and converted into an electric signal. As a solid-state image sensor,
Besides the CCD line sensor, a MOS type or photodiode array can be used as well.

In the DNA nucleotide sequencer of the present invention, a high-intensity light emitting diode is used as an irradiation excitation light source.
When used as an irradiation excitation light source for fluorescence, it is necessary to solve the following problems. Changes in emission wavelength due to changes in band gap with temperature, and changes in emission efficiency due to changes in temperature. Since the fluorescence intensity changes when the above-mentioned and occur,
In a DNA nucleotide sequencer that uses fluorescence intensity as sample information, it is necessary to stabilize the above and.

FIG. 3 is a characteristic diagram showing the relationship between the wavelength (nm) and the emission intensity at each temperature of the high brightness light emitting diode. FIG. 4 is a characteristic diagram showing a change in emission peak wavelength of the high-intensity light emitting diode with temperature. As is clear from FIG. 4, the peak wavelength λp of the high brightness light emitting diode is 0.1
It changes at a rate of 4 nm / ° C. FIG. 5 is a characteristic diagram showing changes in temperature and forward current of the high brightness light emitting diode. As is clear from the characteristics shown in FIG. 5, the forward current of the high-intensity light emitting diode increases as the temperature rises.

Therefore, it is preferable to use the stabilizing circuit of the high brightness light emitting diode chip as shown in FIG. The high brightness light emitting diode chip 1 is a Peltier device 19
And a heat sink 21 is connected to the Peltier element 19. The high-intensity light emitting diode chip 1 is connected to the constant current power supply 2, and a current detector 4 is provided between the high-intensity light emitting diode chip 1 and the constant current power supply 2. The forward current detection value in the current detector 4 is the differential amplifier 6
On the other hand, the temperature setting value of the variable voltage source 8 is also input to the differential amplifier 6. The differential amplifier 6 compares the forward current detection value and the temperature setting value, and the difference is fed back to the Peltier element 19 by the Peltier controller 10 to keep the temperature of the high-intensity light emitting diode chip 1 constant. Specifically, as an adjusting method, first, the high-intensity light emitting diode chip 1 is caused to emit light, and the wavelength is measured. Then
Adjust the temperature setting to give the desired wavelength. afterwards,
The Peltier controller 19 drives the Peltier element 19 to keep the temperature of the high-intensity light emitting diode chip 1 constant. Thus, the wavelength and output of the high-intensity light emitting diode chip 1 can be stabilized. Since the output of high-intensity light-emitting diodes varies depending on each high-intensity light-emitting diode, the output of the whole high-intensity light-emitting diode group can be made constant by integrating each temperature control system with each high-intensity light-emitting diode. .

The emission wavelength of the high brightness light emitting diode is 550
Is about 630 nm. It is preferable to change the emission wavelength according to the fluorescence wavelength of the fluorescent label used. For example, when Texas Red is used as the fluorescent label, since the center wavelength of the fluorescence emitted from this fluorescent label is 615 nm, it is preferable to use a high brightness light emitting diode that emits light in the wavelength band of 590 to 595 nm. Therefore, from the characteristic diagram of FIG. 3, it can be understood that it is preferable to control the temperature so as to maintain each high-intensity light emitting diode at 15 ° C. to 25 ° C.

FIG. 7 is a schematic enlarged perspective view of the gradient index lens array 3. The gradient index lens used in the present invention is also called a selfoc lens, and is a cylindrical lens 9 having a refractive index distributed in the radial direction. When a fluorescent image is formed in close contact with the gradient index lens, a uniform signal level can be obtained between the center and the edge. As a result, S /
The uniformity of N is improved, and it becomes possible to read the base length in the lane around the migration plate as in the vicinity of the center. When the gradient index lenses are arranged in an array, adjacent lens images overlap each other, and a 1: 1 erect image forming system is obtained in the lens arrangement area. The SELFOC lens used in the present invention is, for example, commercially available under the product code of SLA-20B (F0.96) and has a diameter of about 1 mm and a length of about 12 mm.
It is about 13 mm. The SELFOC lens can also be used in a one-step array. However, in the present invention, this SELFOC lens is combined into two stages, one above and one below, and a total of 400 lenses are used per stage to form a lens array. The reason why the SELFOC lenses are used in a combination of two stages, upper and lower, is to increase the amount of light taken in the lens to increase the resolution.
By combining the SELFOC lenses in the upper and lower two stages, the aperture ratio of the entire lens system increases, and it becomes possible to detect even weak fluorescence. Therefore, if desired, it is possible to stack the SELFOC lenses in three or more stages. Needless to say, the lenses 9 are supported by a suitable housing or holder in order to bundle the lenses 9 to form the lens array 3. In the present invention, a camera lens which has been conventionally used can be used instead of the SELFOC lens.

FIG. 8 is a partially cutaway perspective view of an example of an assembly of the fluorescence detecting means 2 shown in FIG.
As shown in FIG. 8, the lens array 3 is sandwiched by a suitable fixing plate 11. One of the fixing plates is fixed to the filter mount 13. Filter mount 1
A two-layer filter 5 is mounted inside the unit 3. A CCD mount 15 is closely attached adjacent to the filter mount 13. CCD inside the CCD mount 15
A line sensor 7 is provided. The CCD heats up due to driving, dark current increases, and noise is generated, which causes S
The / N ratio may be lowered. Therefore, CC
Behind the D-line sensor 7, a soaking zone 17 is closely attached, and an electronic cooling device 19 such as a Peltier element is arranged at the soaking zone 17. CCD by Peltier element 19
It is preferable to maintain the line sensor at a temperature within the range of 10 ° C to 15 ° C. Further, in order to improve the heat dissipation characteristics of the electronic cooler 19, a heat sink 21 is fixed. The soaking zone 17 and the electronic refrigerator 19 are held by a cooling mount 23. CCD is generally CC
Although it is mounted on the D-board and used, the board portion is omitted and not shown in FIG. 8 and FIG. 9 below.

Referring to FIG. FIG. 9 is a schematic sectional view showing a state in which the excitation light source 1 and the fluorescence detection means 2 are arranged in the DNA base sequence determination device. Electrophoresis plate 7 held vertically
The excitation light source assembly 1 is arranged on one side, and the fluorescence detection means assembly 2 is arranged on the other side with 4 interposed therebetween. The excitation light source assembly 1 and the fluorescence detection means assembly 2 are preferably arranged on the same axis and face each other. As with the CCD line sensor 7, the high-intensity light-emitting diode chip has a soaking zone 17 closely attached to the back thereof.
7, a Peltier element 19 is arranged, and a heat sink 21 is fixed in order to improve the heat radiation characteristic of the Peltier element 19. The distance from the laser beam incident position 76 of the migration plate to the front end of the SELFOC lens array is not particularly limited, but is about 15 mm in the embodiment of the present invention. Further, the distance from the rear end of the Selfoc lens array to the surface of the CCD line sensor 7 is not particularly limited, but is about 15 mm in the embodiment of the present invention. The distance from the laser light incident position 76 of the migration plate to the front end of the SELFOC lens array and the CCD from the rear end of the SELFOC lens array
The distance to the surface of the line sensor 7 may be the same or different. However, it is preferred that they are the same. This is because the SELFOC lens has a magnification of 1 and it is preferable that the air gaps in front of and behind the lens are the same.

FIG. 10 is a block diagram of a signal processing system including a CCD circuit. CCD line sensor 7 is CCD circuit 4
Driven by 0. The content itself of the CCD circuit 40 is almost the same as that of a known and commonly used CCD drive circuit. That is, it includes a timing controller, a timing generator circuit, a multiplexer, and the like. The analog output received by the CCD line sensor 7 and processed by the CCD circuit 40 enters the H8 board 42. This analog output is amplified by an amplifier 44, filtered by a low pass filter 46 to remove a noise component, and converted into a digital signal by an A / D converter 48. This digital signal is composed of a memory 50 and a CPU 52. Is processed by. The H8 board can also be connected to a host computer 54. The H8 board 42 also performs I / O of the Peltier device controller 56. A signal from a thermistor (not shown) controls the Peltier device controller 56.
Turns the drive of the Peltier device 19 “ON” / “OFF”. The CCD circuit can include a CCD picture signal integration circuit. This integration circuit not only reduces noise, but also allows pixel adjustment. For example, if the CCD output is 63.5 μm / pix, it is integrated by 4 pixels (pix) to 254 μm / pix, and the result after 4 pix integration is output.

Refractive index distribution type lens array 3, filter 5
And it is preferable that the CCD line sensors 7 all have the same length. These may have the same length as the lateral length of the migration plate 74, that is, the length from the left end to the right end of the migration plate 74, or the length from the migration path at the right end to the migration path at the left end. The length may be slightly shorter than the width of the electrophoretic plate with a smile allowance.

The CCD line sensor 7 is a linear array of CCDs in the horizontal direction, and is also used in a photoelectric conversion unit of an OCR or FAX scanning system. The type of CCD used in the present invention is not particularly limited. For example,
TCD109AC (PIX65 × 65 μm) or the like can be preferably used. The vertical width of the CCD line sensor (that is, the width in the migration direction) is not particularly limited, but is generally 85.
It is preferably not more than μm. The initial stage of migration is DN
Since the A fragment has good separability, it is easy to detect the fragment, but at the final stage of electrophoresis, the DNA fragment becomes poor in separability and the fragments migrate collectively. If the vertical width of the CCD line sensor is 85 μm or less, even if the DNA fragments collectively migrate, each fragment can be detected individually. Also, if the CCD line sensor is used in one stage, it becomes a one-dimensional sensor, but if it is used in a stack of two or more stages, it can be used as a two-dimensional area sensor.

In the embodiment shown in FIG. 2, the light emitted from the high-intensity light emitting diode chip 1 is directly incident on the electrophoretic plate, and one chip irradiates four electrophoretic paths. In the embodiment shown in FIG. 11, the light emitted from the high-intensity light-emitting diode chip 1 is uniformly diffused laterally by the prism 60 and the cylindrical lens 62, and one chip irradiates four migration paths. You can also The reason why the cylindrical lens 62 is used in combination with the prism 60 is that the spread light emitted from the LED is condensed in the migration direction on the sample to increase the irradiation density. By using this combination, it is possible to effectively irradiate only the inside of the detection area.

As shown in FIG. 12A, the lens 6
4 used alone, high brightness LED chip 1
Light from is strong in the center and weak in the surroundings. as a result,
The illuminance distribution on the irradiation surface of the migration plate becomes non-uniform, resulting in an error in the measurement results. On the other hand, as shown in FIG. 12B, when the prism 60 is used, the light from the high-intensity light emitting diode chip 1 is collimated by the prism and the illuminance distribution on the irradiation surface of the migration plate becomes uniform. . As a result, highly reliable measurement results can be obtained.

FIG. 13 is a partial schematic side view of another embodiment of the DNA nucleotide sequencer of the present invention. Unlike the embodiment shown in FIG. 1, both the excitation light source 1 and the fluorescence detection means 2 are arranged on the same side of the migration plate 74. In the embodiment shown in FIG. 13, the excitation light source 1 is made incident by inclining the excitation light source 1 by an angle θ with respect to the normal line to the migration plate 74, and the fluorescence from the sample is similarly inclined by the angle θ. It is detected by the fluorescence detecting means 2.

[0026]

As described above, according to the present invention,
Instead of the conventional laser light source, an inexpensive high-intensity light emitting diode is used as the excitation light source. The high-brightness light emitting diode has a longer life than the laser, and the device can be downsized significantly. The high-brightness light emitting diodes cannot be used as an excitation light source as they are because of variations in output due to the high-brightness light-emitting diodes. However, in the present invention, the high-brightness light-emitting diodes are provided with a temperature control mechanism to achieve high brightness. The output of the light emitting diode group can be made constant, and it can be used as an excitation light source of the DNA base sequencer.

[Brief description of drawings]

FIG. 1 is a partial schematic perspective view of an example of a DNA base sequence determination device of the present invention.

FIG. 2 is a plan view of the device shown in FIG.

FIG. 3 is a characteristic diagram showing a relationship between wavelength and intensity at various temperatures of a high brightness light emitting diode.

FIG. 4 is a characteristic diagram showing a relationship between a temperature and a peak wavelength of a high brightness light emitting diode.

FIG. 5 is a characteristic diagram showing a relationship between temperature and forward current of a high-brightness light emitting diode.

FIG. 6 is a circuit diagram of an example of a temperature control mechanism of a high brightness light emitting diode.

FIG. 7 is a schematic enlarged perspective view of a gradient index lens array.

FIG. 8 is a partially cutaway perspective view of an example of an assembly of fluorescent detection means.

FIG. 9 is a schematic cross-sectional view showing an example of a state in which the assembly of the fluorescence detection means and the assembly of the excitation light source are arranged in the DNA base sequence determination device.

FIG. 10 is a block diagram of a signal processing system including a CCD circuit.

FIG. 11 is a schematic perspective view of an embodiment in which a prism and a cylindrical lens are used together with a high brightness light emitting diode in the excitation light source of the present invention.

FIG. 12 is a schematic diagram showing an illuminance distribution of a high-intensity light emitting diode, (A) showing an illuminance distribution when using a conventional lens system, and (B) showing an illuminance when using a prism according to the present invention. The distribution is shown.

FIG. 13 is a partial schematic perspective view of another example of the DNA nucleotide sequencer of the present invention.

FIG. 14 is a schematic configuration diagram of a DNA base sequence determination device disclosed in Japanese Patent Laid-Open No. 63-21556.

[Explanation of symbols]

 1 High-Brightness Light Emitting Diode 2 Fluorescence Detection Means 3 Gradient Index Lens Array 5 Filter 7 CCD Line Sensor 9 Gradient Index Lens

Claims (5)

[Claims] 1. A vertically-held flat-plate gel electrophoresis means having a plurality of migration paths for fluorescently labeled DNA fragments, an excitation light source for irradiating the migration paths of the electrophoresis means with excitation light, and the excitation. In a DNA base sequencer comprising a fluorescence detection means for detecting fluorescence generated from a fluorescence-labeled DNA fragment irradiated with light and converting it into an electric signal, the excitation light source is a high-intensity light emitting diode (LED), The fluorescence detecting means is composed of a gradient index lens array, a filter and a CCD line sensor, and the high-intensity light emitting diode is connected to a temperature control circuit having an electronic cooler. apparatus. 2. The apparatus of claim 1, wherein the electronic refrigerator is a Peltier device. 3. The device according to claim 1, wherein a prism and a cylindrical lens are used together with the high brightness light emitting diode. 4. A high-intensity light emitting diode is arranged on one side of the electrophoretic plate, and a fluorescence detecting means is arranged on the other side of the electrophoretic plate, and the high-intensity light emitting diode and the fluorescence detecting means sandwich the electrophoretic plate. The apparatus of claim 1 facing directly at. 5. The apparatus according to claim 1, wherein both the high-intensity light emitting diode and the fluorescence detecting means are arranged on the same side of the electrophoresis plate.