JP2000074835A - DNA base sequencer - Google Patents

Abstract

(57) Abstract: Provided is a DNA base sequencer having a novel mechanism for making the wavelength of excitation laser light generated from a laser diode (LD) constant. SOLUTION: Gel electrophoresis means having a migration path for a fluorescence-labeled DNA fragment, a laser diode light source for light excitation for irradiating a laser light to the migration path of the electrophoresis means, and DNA irradiated by the laser light In a DNA base sequencer comprising a CCD sensor for detecting fluorescence generated from a fragment and converting it into an electric signal, a laser diode light source and an electrophoresis means are provided between the laser diode light source and the gel electrophoresis means. A short-pass filter is provided on the optical axis.

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 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. 5 is a schematic diagram illustrating the configuration of the apparatus. In the apparatus shown in FIG. 5, the laser light emitted from the light source 70 is reflected by the mirror 72, and irradiates a predetermined point in the gel of the electrophoresis plate 74 horizontally and horizontally. When the fluorescently labeled DNA fragment 76 that has migrated through the gel passes through the irradiated area, fluorescence is sequentially emitted from the 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.

The apparatus shown in FIG. 5 uses an image intensifier camera as a light receiving system. The image intensifier camera is not only an extremely expensive optical apparatus but also a relatively large apparatus. . For this reason, the size of the entire electrophoresis apparatus has to be increased.

Further, a gas laser light source such as an argon ion laser or a helium-neon (He-Ne) laser is used as a light source. These laser light sources are generally very expensive. Furthermore, the DNA sequencer of the type that irradiates excitation light from the gel side has the following disadvantages.
Since the laser beam of the excitation light cannot be narrowed down over a long range, the spatial resolution is limited. In order to solve this, the beam was expanded elliptically in the migration direction to improve the spatial resolution at the pixels of the sensor. However, gas lasers often have a circular cross-sectional shape, and the beam is expanded elliptically. Irradiating optical system becomes complicated.

[0009] In order to solve these problems, the present inventors have first used a DNA line sensor which uses a CCD line sensor as a light receiving system and a laser diode (hereinafter referred to as "LD") as an excitation light source. We invented and applied for a base sequence determination device (Japanese Patent Application No. 8-308764). L
The beam divergence angle of the excitation light by D is 7.5 × 37 (de)
g). As is clear from these characteristic values, the emission characteristics of the laser diode generate a substantially elliptical beam. Therefore, unlike a gas laser that generates a beam having a circular cross section, a laser diode does not require special optical means for making the beam elliptical. Further, the CCD line sensor is not only significantly smaller than an image intensifier camera but also inexpensive. Similarly, LDs are relatively small compared to gas lasers, and are very inexpensive. Therefore, by using a CCD line sensor as a light receiving system and using an LD as an excitation light source, not only the size of the entire DNA base sequencer could be dramatically reduced, but also the cost could be greatly reduced. .

However, as a result of further research, L
The oscillation wavelength of D is unstable, and it has been found that it is relatively difficult to stably obtain a desired specific wavelength. For this reason, an attempt has been made to keep the current of the LD constant by an automatic output control mechanism (APC), cool the LD with a Peltier element, and maintain the LD at a constant temperature of 20 ° C. Thereby, the wavelength of the excitation light generated from the LD can be controlled within 367 ± 1 nm.

However, even with the use of the LD whose current and temperature are controlled as described above, fluorescence having a wavelength near 367 nm of the excitation light wavelength cannot be clearly separated, which is a major cause of measurement errors. Was.

[0012]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a DNA base sequencer having a novel mechanism for keeping the wavelength of an excitation laser beam generated from an LD constant.

[0013]

The above object is achieved by providing a short-pass filter between an LD light source and an electrophoretic means.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a partial schematic configuration diagram of a DNA base sequence determination apparatus according to the present invention. As shown in FIG. 1, in the apparatus of the present invention, an LD1 is used instead of a gas laser as an excitation light irradiation light source. As this LD, for example, an LD that is generally commercially available under the trade name HL6319G from Hitachi, Ltd. can be used. The emission wavelength of this LD is 637 nm and the output is 7
mW. The beam spread angle of the excitation light is 7.5x
37 (deg). As is apparent from these characteristic values, the light emission characteristic of the LD generates a substantially elliptical beam. Therefore, unlike a gas laser that generates a beam having a circular cross section, the LD does not require special optical means for making the beam elliptical. LDs having specifications other than those described above can of course be used in the apparatus of the present invention.

The excitation light emitted from the LD 1 is focused on a predetermined position of the electrophoretic means 5 by the condenser lens 3. In the apparatus of the present invention, the short-pass filter 2 is provided between the condenser lens 3 and the electrophoretic means 5. The location of the short-pass filter 2 is not limited to the illustrated position.
It may be between D1 and the condenser lens 3. The short-pass filter 2 has a function of cutting out the long-wavelength excitation light from the excitation laser light generated from the LD 1 and transmitting only the excitation light having a desired wavelength of 637 nm. Actually, the short-pass filter 2 has a function of transmitting 90% or more of the excitation light having a desired wavelength of 637 nm and transmitting only about 0.01% of the excitation light having a longer wavelength.

As a result of intensive studies by the present inventors, it has been discovered that commercially available LDs generate excitation light having various wavelengths in addition to excitation light having a wavelength of 637 nm. Of such excitation light, excitation light having a wavelength longer than 637 nm (for example, excitation light having a wavelength of 650 nm) becomes stray light, which makes it difficult to separate from fluorescence having a wavelength of 650 nm, which may cause a measurement error. is there. Accordingly, it is preferable that only the excitation light having a wavelength of 637 nm or less is transmitted through the short-pass filter 2 and that the excitation light having a longer wavelength is cut off.

Although the electrophoresis means 5 shown in FIG. 1 is a single hollow capillary, it may be a plurality of hollow capillaries or a conventional flat electrophoresis plate. DN in which a plurality of hollow capillaries are arranged on the same straight line and a laser beam is irradiated
The A-base sequencer is disclosed in Japanese Patent Application Laid-Open No. 5-72177. A DNA base sequencer using an LD as an excitation light source and a flat electrophoresis plate as an electrophoresis means is disclosed in Japanese Patent Application No. 8-308764 by the present applicant.

When excitation light is applied to the fluorescence-labeled DNA fragment sample inside the electrophoresis means 5, fluorescence of a specific wavelength is generated from the fluorescent label used. For example, D
FITC was used as a fluorescent label for labeling NA fragments.
(Fluorescein isothiocyanate), EITC (eosin isothiocyanate), TMRITC (tetramethyl rhodamine isothiocyanate), XRITC (substituted rhodamine isothiocyanate) and the like are used. Other fluorescent labels can of course be used.

The fluorescent light generated from the fluorescent label is collected by the fluorescent light collecting system 6. The fluorescent light is first condensed by the first lens 7, converted into parallel light, and passes through the long-pass filter 9. The long-pass filter 9 has a wavelength of 65
It is used to transmit only fluorescent light having a wavelength of more than 0 nm and to cut off stray light having a wavelength of less than 650 nm (for example, scattered light of excitation light having a wavelength of 637 nm) that is incident together with the fluorescent light.

The fluorescent light transmitted through the long-pass filter 9 is focused again by the second lens 11. The focal point is, for example, approximately the center of the opening 15 of the slit 13 in FIG. The focused fluorescence is reflected by the mirror 17 and is incident on the grating 18, is reflected again by the grating 18, and finally is incident on the CCD sensor 20.
Grating 18 used in the device shown in FIG.
Is an optical member that performs light separation, wavelength selection, or light polarization by using light diffraction, and has a periodic uneven structure on a flat or concave substrate. A diffraction grating having another structure can be used as the grating 18.

The CCD sensor 20 is known to those skilled in the art.
The CCD sensor 20 used in the device of the present invention is, for example,
In use, it is cooled to, for example, −10 ° C. by a conventional means (for example, a Peltier device) known to those skilled in the art.

The fluorescent light detected by the CCD sensor 20 is sent to a signal processing circuit 22, and the wavelength of the detected fluorescent light is detected corresponding to the pixel position, and subjected to signal processing known to those skilled in the art such as A / D conversion. The data are converted into A, T, C, and G, respectively, and output by an appropriate data output means 24 (for example, a CRT and / or a printer) to determine the base sequence of the DNA fragment.

FIG. 2 is a characteristic diagram showing the effect of using the short-pass filter 2. FIG. 2A is a characteristic diagram showing a detected waveform and Raman luminance when electrophoresis is performed without using the short-pass filter 2. As shown in the figure, an LD sideband having a wavelength of about 650 nm is detected, and fluorescence having a similar wavelength is not separated,
Raman luminance of the peak near the wavelength of about 800 nm is about 40in.
t. On the other hand, FIG.
When the electrophoresis is performed using the short-pass filter 2 as shown in FIG.
Not only is the fluorescence of 50 nm clearly separated, but the Raman luminance of the peak near the wavelength of about 800 nm also increases to 100 int. Therefore, it can be understood that the effect of increasing the Raman luminance by using the short-pass filter 2 is about 2.5 times. The reason why the excitation light having the wavelength of 637 nm is simultaneously detected is that a part of the excitation light having this wavelength passes through the long-pass filter 9 and is received as stray light by the CCD sensor. However, stray light of this wavelength has a wavelength of 650.
nm's original fluorescence,
It does not adversely affect the measurement results.

FIG. 3 shows the DN according to the present invention shown in FIG.
FIG. 2 is a schematic front view of a partially cut-away portion of the A-base sequencer.
One end of the capillary 5 is immersed in the buffer solution in the lower buffer tank 60, and the other end is immersed in the buffer solution in the upper buffer tank 62-1 of the sample tray 62 of the sample supply unit 61. A high voltage is applied to the lower buffer tank 60 and the upper buffer tank 62-1 from the high voltage power supply 63. For example, a negative voltage of −15 kV is applied to the lower buffer tank 60. The upper buffer tank 62-1 is grounded. In order to keep the temperature of the gel electrolyte in the capillary 5 constant during the electrophoresis, a heater unit 64 for controlling the temperature is provided behind the capillary 5. A window is opened at a predetermined position of the heater unit 64, and the fluorescent light condensing system 6 shown in FIG. The capillary 5 is positioned at this window so as to face the fluorescent light collecting system 6.

FIG. 4 is a top view of the sample tray 62. As shown, the sample tray 62 is disk-shaped, and when mounted on the sample supply unit 61,
It is configured to be able to rotate clockwise and counterclockwise.
Reference numeral 62-1 denotes an upper buffer tank, and reference numeral 62-2 denotes a "garbage reservoir" for discarding the gel electrolyte and the DNA sample in the capillary after the electrophoresis. Symbol 62-3
Is a spare upper buffer tank, and reference numeral 62-4 is a spare "garbage reservoir". The fluorescence-labeled DNA fragment sample is provided in holes indicated by reference numerals 62-5 to 62-n.
The upper buffer tank, the “garbage reservoir” and the fluorescent-labeled DNA fragment sample are each labeled with a plastic tube.
It can also be used in a mode of being inserted into each hole indicated by -1 to 62-n.

Referring back to FIG. When the gel electrophoresis is completed, the sample supply unit 61 is lowered, and the capillary 5 is extracted from the upper buffer tank 62-1. Then
The sample supply unit 61 rotates by a predetermined angle, then rises again, and inserts the capillary 5 into the “garbage reservoir” 62-2. When one end of the capillary 5 is closed by the stopper 65 and the piston of the syringe 66 for gel electrolyte injection is lowered, the gel electrolyte and the DNA fragment sample inside the capillary 5 are "garbage reservoir" 62-2 from the open other end.
Is discharged into Next, in this state, the gel electrolyte injecting syringe 66 is filled with fresh gel electrolyte, and the piston is lowered, whereby the empty capillary 5 is filled with fresh gel electrolyte. Thereafter, the sample supply unit 61 descends, and extracts the capillary 5 from the “garbage reservoir” 62-2. Next, the sample supply unit 61 rotates by a predetermined angle, and then rises again. The capillary 5 is inserted into a tube filled with a DNA fragment sample in a predetermined position of the section 62-m, and the syringe 66 for gel electrolyte injection is inserted. Aspirate the sample by raising the piston. After that, the sample supply unit 61 is lowered, rotated to the position of the upper buffer tank 62-1 and then raised, and the other end of the capillary 5 is connected to the upper buffer tank 6-1.
2-1 and immersed in the upper buffer solution. This completes the sample loading. When the stopper 65 is released and a voltage is applied from the high-voltage power supply 63, a new gel electrophoresis is started. These series of operations can be programmed and stored in an appropriate memory, and all can be automatically performed. Such automated means are known to those skilled in the art.

[0027]

As described above, according to the present invention,
By arranging a short-pass filter between the LD light source and the gel electrophoresis means, unnecessary side bands generated from the LD light source can be cut, and as a result, the fluorescence separation ability is dramatically improved. In addition, the Raman luminance can be increased 2.5 times.

[Brief description of the drawings]

FIG. 1 is a partial schematic configuration diagram of a DNA base sequence determination device according to the present invention.

FIG. 2 is a characteristic diagram showing an effect of using a short-pass filter. (A) is a characteristic diagram showing a detection waveform and Raman luminance when electrophoresis is performed without using a short-pass filter, and (b) is a detection waveform when electrophoresis is performed using a short-pass filter. It is a characteristic view showing Raman luminance.

FIG. 3 shows DNA using a single capillary according to the present invention.
It is a partial notch outline front view of a base sequence determination apparatus.

FIG. 4 is a top view of a sample tray used in the apparatus shown in FIG.

FIG. 5 shows a D disclosed in JP-A-63-21556.
FIG. 2 is a schematic configuration diagram of an NA base sequencer.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Laser diode 2 Short pass filter 3 Condensing lens 5 Electrophoretic means 7 First lens 9 Long pass filter 11 Second lens 13 Slit 15 Slit opening 17 Reflection mirror 18 Grating 20 CCD sensor 22 Signal processing circuit 24 Data output means 60 Lower buffer tank 61 Sample supply unit 62 Sample tray 63 High voltage power supply 64 Heater unit 65 Stopper 66 Syringe for gel injection

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G043 AA03 AA04 BA16 CA03 DA02 EA01 EA03 EA19 GA08 GB07 HA01 HA02 JA03 JA04 KA05 LA03 MA01 MA11

Claims (3)

[Claims] 1. A gel electrophoresis means having a migration path for a fluorescently labeled DNA fragment, a laser diode light source for light excitation for irradiating a laser beam to the migration path of the electrophoresis means, and a laser beam. A DNA base sequencer comprising a CCD sensor that detects fluorescence generated from a DNA fragment and converts the fluorescence into an electric signal, wherein a laser diode light source and an electrophoresis means are provided between the laser diode light source and the gel electrophoresis means. 3. A DNA base sequence determination device, wherein a short-pass filter is provided on the optical axis. 2. A fluorescent light condensing lens is further provided between the laser diode light source and the short pass filter on the optical axis of the laser diode light source and the short pass filter. The DNA base sequencer according to claim 1. 3. The gel electrophoresis means is a single hollow capillary, a long-pass filter is further provided between the gel electrophoresis means and the CCD, and a first pass filter is provided before the long-pass filter. The second lens is disposed after the long-pass filter, and the second lens is disposed after the long-pass filter.
3. The method according to claim 1, wherein the fluorescent light transmitted through the lens is passed through the opening of the slit and is incident on the grating by the reflecting mirror, and the fluorescent light reflected by the grating is received by the CCD sensor. The DNA base sequencer according to claim 1.