JPH08327598A - Electrophoretic device - Google Patents

Abstract

PURPOSE: To provide an electrophoretic device that detects fluorescence-labeled DNA at high sensitivity. CONSTITUTION: This electrophoretic device, which separates and detects samples of nucleic acid labeled with a fluorescent substance, includes an electrophoretic part 2 containing a polyacrylamide gel in which the samples of nucleic acid migrate, a laser beam source 1 which emits exciting light for exciting the fluorescent substance as it is applied to the samples of nucleic acid migrating in the polyacrylamide gel, and a light detection means 10 for detecting fluorescence caused by the application of the exciting light 4, the wavelength of the exciting light being 530nm or more so that the light detection means 10 detects fluorescence in a wavelength range where light emission from the gel is weak. Therefore, DNA labeled with a trace amount of fluorescent substance can be detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to an electrophoretic device for determining a DNA base sequence or detecting a fluorescently labeled DAN such as a DNA probe.

[0002]

2. Description of the Related Art Conventionally, radioactive phosphorus (
A method has been used in which a DNA fragment is labeled with ³² P) and the pattern after electrophoretic separation is transferred to a photographic filter by autoradiography. Recently, FITC (fluorescein isothiocyanate) (fluorescein isot), which is used for labeling fluorescent substances and proteins excited by ultraviolet rays, is used.
hiocyanate), TRITC (tetramethylrhodamine isothiocyanate) (tetramethy
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]

The difficulty of such a DNA detection method using a fluorescent label is that sufficient detection sensitivity cannot be obtained. That is, scattered light and fluorescence emitted from the gel are observed in addition to the fluorescence emitted from the fluorescence-labeled DNA which is subjected to light irradiation during the electrophoresis of the fluorescence-labeled DNA or during migration to detect the DNA. Therefore, there is a problem that the weak light emitted from DNA cannot be detected. An object of the present invention is to overcome the above problems and provide an electrophoretic device for detecting fluorescently labeled DNA with high sensitivity.

[0004]

[Means for Solving the Problems] The above-mentioned object is that the wavelength of the excitation light source is 540 nm or more by studying the spectral characteristics of the fluorescence from the gel, which is harmful in measurement, investigation of the cause, and careful selection of the excitation light source and the fluorescent dye. This is achieved by reducing the gel fluorescence and relatively increasing the fluorescent dye emission as a laser of.

[0005]

[Effect] Ethenoadenosine, FITC, TRITC and Texas Red used as fluorescent substances for labeling
The optimum excitation wavelength and the maximum fluorescence emission wavelength of (3
00nm, 410nm), (490nm, 520n
m), (550 nm, 580 nm), and (580n
m, 605 nm). As a laser light source that is convenient for exciting these dyes and has a relatively large output, YAG
(4th harmonic 265 nm) or Ar (488 nm, 514.5)
nm) laser is used. On the other hand, the light emission from the gel greatly changes depending on the excitation wavelength and the emission wavelength. FIG. 1 shows the dependence of gel emission intensity on excitation light, and the emission wavelength is made to coincide with the emission wavelength of each dye. That is, 101
Is 520 nm, 102 is 580 nm, and 103 is 605 n
It is the light emission of m. 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 is caused by impurities in the gel material,
Some are caused by the polymerization of acrylamide.
The latter is large when the excitation wavelength is short, but decreases as the wavelength becomes longer, and becomes very small at the excitation wavelength of 530 to 540 nm or more. Therefore, as an excitation light source,
Using a laser with a wavelength of 0 nm or more, the emission wavelength is 6
By labeling the DNA with a fluorescent dye having an emission wavelength of around 00 nm or longer, the DNA fragment can be detected with high sensitivity.

[0006]

Embodiment An embodiment of the present invention will be described with reference to FIG. The measuring device includes a light source 1, an electrophoretic 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 constant distance from the migration start point of the migration plate. In this embodiment, the laser light is incident on the gel from a direction parallel to the gel plane, that is, from the gel side surface, but the laser light may be incident obliquely forward or backward to scan. The sample is adjusted as follows. One end of the sample DNA is labeled with Texas Red or TRITC fluorophore and the other end is terminated with an adenine base (A) to prepare a fragment group {A} of various lengths. 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 electrophoresed. The longer the DNA fragment is, the slower the DNA fragment migrates, and the shorter the DNA fragment migrates faster. Therefore, the DNA fragments are separated into bands as shown in FIG.

After passing through the filter, the fluorescent light is imaged as a fluorescent image on the image amplifier 7 using a condenser lens, amplified, detected by the photodiode array 8 and processed by the computer 12. In this embodiment, the area between a and b of the irradiated portion of the racer is detected, and several migration paths cross this area. Focusing on one migration path, the fluorescence signal increases each time the fluorescent label DNA passes through the irradiation line, so 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 into each migration path is known, the base sequence can be determined by sequentially reading the signals that appear with time.

FIG. 3 shows FITC, 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 an excitation light source, TR
543nm He-Ne laser, Tex for ITC
A 567 nm krypton laser is optimal for as Red. When these are used, the emission intensity is Texas.
The order is Red, FITC, TRITC. FITC
The strength varies depending on the pH of the solvent, but it is almost Te.
It can be evaluated as equivalent to xas Red. However, the fluorescence intensity normalized by the background light intensity from the gel is very different. FIG. 4 shows the strength of each phosphor when the background light of the gel is 1. The concentration of the phosphor is 1 nmole / 1.

Although the absolute amount of fluorescence emitted from Texas Red excited at 543 nm is smaller than that of FITC, the amount of gel fluorescence is smaller (see FIG. 1), so that the relative intensity is larger than that of FITC and high sensitivity can be 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 inexpensive lasers provide higher sensitivity. The actual detection limit also depends on the absolute intensity of the fluorescence. If the fluorescence intensity is high, the fluctuation becomes small, and even a trace amount can be detected.

Generally, the light emission signal from the phosphor is received through the interference filter. The interference filter has a narrow transmission wavelength band and a low transmittance. 54, which has a smaller output than the Ar laser (usually using 10 to 20 mW) as in this embodiment.
When using a 3 nm He-Ne (maximum output 1 mW) laser, it is necessary to devise a light receiving system to increase the amount of received light and 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 sharper the rise of the transmission band of the bandpass filter, the better, but normally the transmittance is 0%.
And the wavelength difference between the wavelength range of the wavelength band and the wavelength range of the transmission band having the transmittance of 70 to 80% or more is 20 to 30 nm or more. A filter was designed using FIG. 4, and a filter having a high transmittance in the region of 595 nm to 620 nm and abruptly decreasing the transmittance on the shorter and longer wavelength sides was used. With this filter, the transmittance at 570 nm is almost 0%.

A krypton laser (568 nm) is used as a light source.
However, it is necessary to use a colored glass filter together with a bandpass 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.

[0012]

According to the electrophoretic device of the present invention, since the fluorescence emission in the wavelength region where the gel emission is small is utilized, it is possible to detect a small amount of the fluorescent labeled DNA with high sensitivity.

[Brief description of drawings]

FIG. 1 is a characteristic diagram showing excitation light dependence of fluorescence intensity generated from gel.

FIG. 2 is a conceptual diagram of a measuring device.

FIG. 3 is an excitation spectrum of various dyes.

FIG. 4 is a characteristic diagram showing emission wavelength dependence of dye fluorescence intensity normalized by gel fluorescence.

[Explanation of symbols]

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 ... Fluorescence receiving system, 11 ... Migration power supply, 12 ... Computer, 13 ... Output device, 14 ... Data chart.

Claims (5)

[Claims] 1. An electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, the electrophoresis section including a polyacrylamide gel on which the nucleic acid sample migrates, and the nucleic acid sample on which the polyacrylamide gel migrates are irradiated. A laser light source that emits excitation light that excites the phosphor, and a photodetector that detects fluorescence generated by irradiation of the excitation light, and the wavelength of the excitation light is 530 nm or more,
The light detecting means includes a bandpass filter, and a wavelength difference between a wavelength at which the transmittance of the bandpass filter is 0% and a wavelength region of a transmission band at which the transmittance is 70% or more is about 20 or more, The electrophoretic device, wherein the light detecting means detects the fluorescence in a wavelength region where the light emission from the polyacrylamide gel is small. 2. An electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, and irradiating the electrophoresis section containing a polyacrylamide gel on which the nucleic acid sample migrates and the nucleic acid sample on the polyacrylamide gel. A laser light source that emits excitation light that excites the phosphor, and a photodetector that detects fluorescence generated by irradiation of the excitation light, and the wavelength of the excitation light is 530 nm or more,
The difference between the wavelength of the excitation light and the maximum emission wavelength of the phosphor is about 25 nm or more, and the light detecting means detects the fluorescence in a wavelength region in which the light emission from the polyacrylamide gel is small. Electrophoresis device. 3. An electrophoresis apparatus for separating and detecting a nucleic acid sample labeled with a fluorescent substance, and irradiating an electrophoresis section containing a polyacrylamide gel on which the nucleic acid sample migrates, and the nucleic acid sample on which the polyacrylamide gel migrates. A laser light source that emits excitation light that excites the phosphor, and a photodetector that detects fluorescence generated by irradiation of the excitation light, and the wavelength of the excitation light is 530 nm or more,
The difference between the wavelength of the excitation light and the maximum emission wavelength of the phosphor is in the range of about 25 nm to about 40 nm, and the photodetection means detects the fluorescence in a wavelength region in which the emission from the polyacrylamide gel is small. An electrophoretic device characterized by: 4. The electrophoretic device according to claim 2 or 3, 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. The electrophoretic device according to claim 2, wherein the ratio of the intensity of the fluorescence to the intensity of the fluorescence emitted from the polyacrylamide gel is 10 or more.