JPH011949A - How to regenerate electrophoresis gels - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for regenerating electrophoresis gels for separating and detecting DNA, RNA, proteins, etc. using fluorescent labels.

[Prior Art] Conventionally, gel electrophoresis has been used to separate DNA and the like. Detection is performed by radioactively or fluorescently labeling DNA and the like. In this case, the labeled sample remains in the gel when the -degree separation measurement is performed, so the separation gel cannot be used repeatedly. For this reason, a gel is prepared each time a measurement is made.

Recently, disposable gels in which gel is retained in a film have been commercially available for use with radioactively labeled samples, making it possible to save time and effort in gel preparation. However, this gel cannot be used for photodetection; fluorescence is
This is because when the gel is irradiated with light, strong fluorescence is emitted from the film section, making it impossible to observe the fluorescence from the fluorescent label raDNA.

[Problems to be Solved by the Invention] It is thought that DNA and RNA base sequencing methods and protein detection methods will shift from conventional methods using radioactive labels to methods using fluorescent labels. However, so far, neither the development of disposable gels suitable for fluorescence methods nor the method of recycling gels has been proposed, and gels must be prepared for each measurement, which is time-consuming and difficult. Ta.

An object of the present invention is to provide an electrophoresis gel regeneration method that eliminates the trouble of preparing a gel for each measurement when using a fluorescent label, and allows repeated use of gels prepared several times. be.

[Means for solving the problem] The above purpose is to use a substance that is easily photodegradable as a fluorescent label, and to irradiate light onto the fluorescently labeled DNA remaining in the gel after the measurement is completed, to change the quality of the fluorescent substance and make it fluoresce. This is achieved by making the substance you want to emit.

[Function] Generally, when a phosphor is irradiated with light, it is excited and emits fluorescence, but at the same time a decomposition reaction proceeds. Ease of decomposition varies depending on the phosphor, but FITC (fluoresceine
1sothiocyanate; excitation wavelength 494n
m, emission wavelength 511 nm), it is extremely easy to decompose. DNA or the like is labeled with these fluorescent substances, and when the fluorescent labels remaining in the gel are exposed to strong light after measurement, they decompose and no longer emit fluorescence. Of course, DNA and the like still exist, but since they are no longer detected by the optical meter, they can be used again as a gel for separation and detection.

[Example] An example of the present invention will be described below with reference to FIGS. 1 and 2. FIG. 1 is an explanatory diagram of the principle of base sequencing, and FIG. 2 is an embodiment of the present invention. In this example, DNA base sequencing is performed using a fluorescent label.

One end is labeled with FITC, and the other end is labeled with adenine (A), titanium (T), and cytosine (C).

Or a group of four fragments (A) ending in guanine (G),
(T), (C), and (a) on a fluorescent gel plate 10
onto separate migration paths. The electrophoretic gel plate 10 is held between gel holding glass plates, and spacers 12 are provided at both ends. A certain area from the starting point of migration migration is irradiated with a laser beam 9 generated by a laser generator 3 for detecting a fluorescent substance, and the fluorescence emitted from the fluorescently labeled DNA 7 passing through the area is filtered through a filter 13. Condensing lens 14. The light is received by a diode array 16 via an image amplifier 15. D.N.
The shorter the length of A, the faster it migrates, so the length of the base can be determined from the luminescence time, and the type of base can be determined from the type of migration path to determine the base sequence. 10 mW as excitation light source 3 for sequencing
An argon laser (488 nm) is used. Typically, electrophoresis is performed for 5 to 6 hours, and sequences of up to about 300 bases are determined. After completion of the electrophoresis, a long length of fluorescently labeled DNA remains between the electrophoresis light spot and the light irradiation detection area.

If the next sample is to be measured in this state, the signals caused by the previous sample will overlap, causing trouble.

Therefore, an argon laser (488 nm) of about IW is focused from the fluorescent substance erasing laser source 4 to a reflection mirror 5.about.1 mm in diameter. Gel 10 from the side direction via movable mirror 6
A laser beam 8 was input into the gel, and the movable mirror 6 was moved and scanned to irradiate the gel surface to decompose the FITC. The electrophoresis plate 10 treated in this manner was reusable. However, in order to apply this method, there were limitations on the decomposition cross-sectional area of the labeled object and the laser intensity.This point will be considered below. Actual decomposition cross section of FITC! When I measured it, it was about 0.5 x 10"' cm2. When the IW argon laser is focused on an area of 1 mm 2 , the photon density is approximately 2.5 × 101 g/mm ”·sec.

Based on the decomposition cross section and photon density, FITC is 0.0 in this optical path.
It decomposes in about 1 second. 1mm for complete disassembly
It is necessary to irradiate about 0.5 seconds per length. Remaining FI
The length of the region where the TC label is desired to be photodecomposed is 200 mm, and if the laser beam 8 is applied at a rate of 2 mm/sec, the time required to irradiate the entire region is 100 seconds. If a cheaper 100 mW laser source is used as a laser source, it will take about 20 minutes for 1000 seconds. The time required for erasing must be within one hour at most. This is because if it takes longer than this, it would be faster to create a new gel plate. If the cross-sectional area of the decomposition is small, it takes a long time to erase, which is inconvenient, and the limit is about IQ-2cm2.In this case, even if an IW laser is used, it takes 20 seconds per 1mm to erase the same way as before. Required and 1 to erase the whole
On the other hand, it is not convenient if the exploded cross section is too large. Normally, the cross-sectional area that emits fluorescence is 10-1g0
A substance with a size of about m2 and a decomposition cross section of 1010-200 will emit an average of 10' fluorescence before it decomposes. If the decomposition cross section is large, the amount of fluorescence emitted before decomposition is small and high sensitivity cannot be obtained.

Under normal DNA separation and detection conditions, it takes 20 to 30 seconds for a DNA band to pass through a 0.5 mm wide irradiation detection area. If the amount decomposed within this time is at most 50%, there will be no problem in detection. For detection, a 5-10 mW argon laser was used at 0.5 mm.
The photon density is approximately 2~
The photon density is 5×10”/m m2・sec.The photon density is l
When Xl0'' pieces/mm 2 ·sec, a substance with a decomposition cross section of 10-19 cm'' decomposes in about 1 second, and it is not preferable for the decomposition cross section to be larger than this.

However, when using a fluorescent labeling compound with a decomposition cross section in the range of 10-19 to 10-22, highly sensitive fluorescence detection and gel reuse by photolysis can be performed.

There are various methods of irradiating light, such as irradiating in a line and sweeping, and irradiating the entire surface using a lamp.

[Effects of the Invention] As described above, according to the present invention, after the state of electrophoretic separation is detected by fluorescence using a fluorescent labeling compound, the fluorescent label remaining in the electrophoresis plate is photolyzed and the gel plate is removed. Enables reuse.

[Brief explanation of the drawing]

FIG. 1 is a conceptual diagram of an embodiment of the present invention, and FIG. 2 is a perspective view of the embodiment of the present invention. 3... Laser for detecting fluorescent substance, 4... Laser for erasing fluorescent substance, 5... Reflecting mirror, 6... Operating mirror. 7...DNA, 8...Laser beam for erasing, 9...
・Laser beam for detection, 10...Gel plate for separation, 11
...Gel holding glass plate, 12...Transparent spacer, 13...Filter, 14...Condensing lens, 15
...Image amplifier, 16...Diode array. 1θ 7th circle

Claims (1)

[Scope of Claims] 1. The gel separation section that electrophoretically separates fluorescently labeled DNA, RNA, or protein and detects it optically is irradiated with light to destroy the fluorescent substance remaining in the electrophoresis gel and generate electricity. A regeneration method for electrophoresis gels that enables reuse of electrophoresis gels. 2. A method for regenerating an electrophoretic gel according to claim 1, characterized in that a substance having a decomposition cross section of 10^-^2^1 cm^2 or more is used as the fluorescent label.