JP2661606B2 - Electrophoresis device - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus for determining a DNA base sequence or detecting a fluorescently labeled DAN such as a DNA probe. [0002] Conventionally, radioactive phosphorus (
^{A method} of labeling a DNA fragment with ³² P) and transferring the pattern after electrophoretic separation to a photographic filter by autoradiography has been used. Recently, FITC (fluorescein isothiocyanate) used for labeling fluorescent substances and proteins excited by ultraviolet light has been developed.
HIOCYANATE), TRITC (tetramethylrhodamine isothiocyanate) (tetramethyrate)
l rhodamine isothiocyanat
e), Texas Red (Sulforhodamin 1
01) A method of detecting a nucleic acid fragment by labeling a nucleic acid with a dye such as (sulforhodamine 101) and detecting the fluorescence emitted by exciting the labeled DNA with an ultraviolet laser or an argon laser (488 nm, 514 nm). . (Nature, 321, 674
(1986)). [0003] A disadvantage of such a DNA detection method using a fluorescent label is that sufficient detection sensitivity cannot be obtained. That is, in addition to the fluorescence emitted from the fluorescently labeled DNA whose DNA is to be detected by irradiating the fluorescently labeled DNA into the gel or during the electrophoresis during the migration, scattered light and fluorescence emitted from the gel are observed. Therefore, there was a problem that weak light emitted from DNA could not be detected. An object of the present invention is to provide an electrophoresis apparatus which can overcome the above problems and detect fluorescently labeled DNA with high sensitivity. [0004] The object of the present invention is to examine the spectral characteristics of fluorescence from a gel which is harmful to the measurement, to investigate the cause, and to carefully select the excitation light source and the fluorescent dye to obtain the wavelength of the excitation light source. Is achieved by using a laser of 540 nm or more to reduce gel fluorescence and relatively increase fluorescent dye emission. [0005] Ethenoadenosine, FITC, TRITC and Texas Red used as fluorescent substances for labeling
The optimal excitation wavelength and the maximum fluorescence emission wavelength of
00 nm, 410 nm), (490 nm, 520 n
m), (550 nm, 580 nm), and (580 n
m, 605 nm). YAG is a laser light source that is convenient for exciting these dyes and has a relatively large output.
(The fourth harmonic 265 nm) and Ar (488 nm, 514.5).
nm) laser is used. On the other hand, the luminescence from the gel changes greatly depending on the excitation wavelength and the emission wavelength. FIG. 1 shows the dependence of the gel emission intensity on the excitation light. The emission wavelength is made to coincide with the emission wavelength of each dye. That is, 101
Are 520 nm, 102 is 580 nm, 103 is 605 n
m emission. This indicates that the longer the excitation wavelength and the emission wavelength become, the smaller the light emission from the gel becomes. The light emitted from the gel emits impurities from the gel material,
Some are caused by polymerization of acrylamide.
The latter is large when the excitation wavelength is short, but decreases as the wavelength increases, and becomes very small at an excitation wavelength of 530 to 540 nm or more. Therefore, 530 to 54 are used as excitation light sources.
Using a laser having a wavelength of 0 nm or more, the emission wavelength is 6
By labeling DNA with a fluorescent dye having an emission wavelength of about 00 nm or more, DNA fragments can be detected with high sensitivity. An embodiment of the present invention will be described with reference to FIG. The measuring device includes a light source 1, an electrophoresis separator 2, and a detector 10
Consists of A 543 nm oscillation He-Ne laser (1 mW) was used as a light source. The electrophoretic separation was performed by electrophoresis using a polyacrylamide plate. After the laser light is narrowed down by a lens, it irradiates a portion at a certain distance from the migration start point of the migration plate. In the present embodiment, the laser beam is incident on the gel from a direction parallel to the gel plane, that is, from the gel side surface. The preparation of the sample is performed as follows. One end of the sample DNA is labeled with a Texas Red or TRITC fluorophore, and fragment groups {A} of various lengths ending with an adenine base (A) at the other end are prepared. Similarly, a fragment group {G} whose other end is G, C or T,
Create {C} and {T}. That is, four types of fragment groups are prepared for each type of DNA whose base sequence is to be determined.
These fragment groups are injected into separate wells 3 and electrophoresis is performed. The longer the DNA fragment, the slower the DNA fragment migrates and the shorter the DNA fragment, the faster the DNA fragment is. Therefore, the DNA fragments are separated into bands as shown in FIG. After the fluorescence passes through the filter, it is formed as a fluorescent image on an image amplifier 7 using a condenser lens, amplified, detected by a photodiode array 8 and processed by a computer 12. In this embodiment, a region between a and b of the irradiated portion of the racer is detected, but this region is crossed by a number of migration paths. Focusing on one migration path, the fluorescent signal increases each time the fluorescent label DNA passes through the irradiation line, so that the time change of the signal for each migration path is as shown in FIG. Since the type (A, G, C or T) of the fragment group injected for each migration path is known, the base sequence can be determined by sequentially reading signals that appear with time. FIG. 3 shows FITC and T used for fluorescent labeling.
It is an excitation spectrum of RITC and Texas Red. The sample concentration is 10 nmole / 1. 488nm argon laser for FITC as excitation light source, TR
543nm He-Ne laser for ITC, Tex
A 567 nm krypton laser is optimal for as Red. When these were used, the emission intensity was Texas
Red, FITC, TRITC. FITC
Varies in strength depending on the pH of the solvent, but almost Te
It can be evaluated as equivalent to xas Red. However, the fluorescence intensity normalized by the background light intensity from the gel differs greatly. FIG. 4 shows the virtue of each phosphor when the gel background light is set to 1. The concentration of the phosphor is 1 nmole / 1. Although the absolute amount of fluorescence emitted from Texas Red excited by 543 nm is smaller than that of FITC, the amount of gel fluorescence is small (see FIG. 1), so that the relative intensity is larger than that of FITC, and high sensitivity is obtained. As is clear from FIG. 4, the emission wavelength is about 595 nm or more and about 640 nm.
In the following, it is clear that the ratio of the intensity of the fluorescence emitted from the phosphor to the intensity of the fluorescence emitted from the gel is 5 or more when excited by 543 nm and 13 or more when excited by 580 nm. He-Ne lasers are much cheaper than argon lasers, and higher sensitivity can be obtained with inexpensive lasers. The actual detection limit also depends on the absolute intensity of the fluorescence. When the fluorescence intensity is high, the fluctuation is small, and a very small amount can be detected. Normally, a light emission signal from a phosphor is received through an interference filter. The interference filter has a narrow transmission wavelength band and a low transmittance. As shown in this embodiment, the output 54 is smaller than that of the Ar laser (usually using 10 to 20 mW)
When using a 3 nm He-Ne (maximum output: 1 mW) laser, it is necessary to improve the light receiving system to increase the amount of received light and to optimize the filter design to prevent the loss of fluorescence. In this embodiment, a bandpass filter having a wide transmission wavelength band and a high transmittance is used. The rise of the transmission band of the band-pass filter is better the steeper, but usually the transmittance is 0%.
And a wavelength difference between the wavelength region of the transmission band where the transmittance is 70% to 80% or more is 20 nm to 30 nm or more. The filter was designed with reference to FIG. 4, a high transmittance was set in the range of 595 nm to 620 nm, and a filter whose transmittance abruptly attenuated on the shorter and longer wavelength sides was used. In this filter, the transmittance at 570 nm is almost 0%. [0011] Krypton laser (568 nm) as the light source
Although higher sensitivity can be obtained by using a color glass filter, it is necessary to use a color glass filter in combination with a band pass filter in order to remove scattered light entering the light receiving system. 57 as a light source
A dye laser, a copper vapor laser, a semiconductor laser, or the like whose wavelength is set to 0 to 580 nm can also be used. According to the electrophoresis apparatus of the present invention, a small amount of fluorescently labeled DNA can be detected with high sensitivity because the fluorescence emission in the wavelength region where the gel emission is small is used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a characteristic diagram showing excitation light dependence of fluorescence intensity formed from a gel. FIG. 2 is a conceptual diagram of a measuring device. FIG. 3 shows excitation spectra of various dyes. FIG. 4 is a characteristic diagram showing emission wavelength dependence of dye fluorescence intensity normalized by gel fluorescence. [Description of References] 1 ... Laser, 2 ... Electrophoresis gel, 3 ... Sample injection well, 4 ... Laser optical path, 5 ... Mirror, 6 ... Lens, 7 ...
Image amplifier, 8: diode array, 9: filter, 10: fluorescent light receiving system, 11: electrophoresis power supply, 12: computer, 13: output device, 14: data chart.

Continuation of front page    (56) References JP-A-63-229365 (JP, A)                 JP-A-63-109370 (JP, A)                 JP-A-59-126246 (JP, A)                 JP-A-61-173158 (JP, A)                 JP-A-60-220860 (JP, A)                 JP-A-61-88155 (JP, A)                 Shokai 63-88747 (JP, U)

Claims (1)

(57) [the claims] 1. In an electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, an electrophoresis section including a polyacrylamide gel on which the nucleic acid sample migrates, and irradiating the nucleic acid sample on the polyacrylamide gel with the fluorescent substance A laser light source that emits excitation light for exciting light, and light detection means for detecting fluorescence generated by the irradiation of the excitation light, wherein the wavelength of the excitation light is 530 nm or more, and the light detection means is a bandpass filter. A wavelength at which the transmittance of the bandpass filter is 0% and a transmittance of 70%
An electrophoretic device, wherein the wavelength difference from the above-mentioned transmission band wavelength region is about 20 nm or more, and the light detection means detects the fluorescence in a wavelength region where light emission from the polyacrylamide gel is small. 2. In an electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, an electrophoresis section including a polyacrylamide gel on which the nucleic acid sample migrates, and irradiating the nucleic acid sample on the polyacrylamide gel with the fluorescent substance A laser light source that emits excitation light that excites light, and light detection means that detects fluorescence generated by the irradiation of the excitation light, wherein the wavelength of the excitation light is 530 nm or more, and the wavelength of the excitation light and the fluorescence The difference from the maximum emission wavelength of the body is about 25
nm or more, and wherein the light detection means detects the fluorescence in a wavelength region where light emission from the polyacrylamide gel is small. 3. In an electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, an electrophoresis section including a polyacrylamide gel on which the nucleic acid sample migrates, and irradiating the nucleic acid sample on the polyacrylamide gel with the fluorescent substance A laser light source that emits excitation light that excites light, and light detection means that detects fluorescence generated by the irradiation of the excitation light, wherein the wavelength of the excitation light is 530 nm or more, and the wavelength of the excitation light and the fluorescence The difference from the maximum emission wavelength of the body is about 25
an electrophoresis apparatus, wherein the fluorescence is in a wavelength range of about 40 nm to about 40 nm, and wherein the light detecting means detects the fluorescence in a wavelength region where light emission from the polyacrylamide gel is small. 4. 4. The electrophoresis apparatus according to claim 2, wherein a ratio of the intensity of the fluorescence to the intensity of the fluorescence emitted from the polyacrylamide gel is 5 or more. 5. 4. The electrophoretic device according to claim 2, wherein a ratio of the intensity of the fluorescence to the intensity of the fluorescence emitted from the polyacrylamide gel is 10 or more.