JPS6375652A - Optically sensitized electrophotoretic gel - Google Patents

Abstract

PURPOSE:To measure the beta-rays from a nucleic acid fragment at a wide solid angle, in an electrophoretic gel for separating the nucleic acid fragment labelled with a radioactive isotope through migration, by mixing a granular or liquid scintillator with a part of the electrophoretic separation gel and detecting the fluorescence from the resulting mixture. CONSTITUTION:An electrophoretic separation gel having nucleic acid fragments 4 labelled with a radioactive isotope inserted therein here and there is held between a pair of glass plates 3 opposed to each other and the beta-rays from the nucleic acid fragments 4 are detected by an electron-multiplier tube 12. At this time, since the intensity of beta-rays is weak, a granular scintillator 20 composed of usual plastic is premixed with the nucleic acid fragments 4 and the beta-rays 7 generated by applying voltage to the gel 2 are allowed to impinge against the scintillator 20 to generate fluorescence 8. Thereafter, a part of beta-rays 7 and the fluorescence 8 are reflected from a reflecting mirror 21 having a film 23 adhered thereto provided behind the gel 2 and allowed to pass through the slits formed to the film 23 and a cylindrical reflecting material 22 to be incident to the electron-multiplier tube 12. By this method, the detection sensitivity of the fragments 4 is made high and beta-rays can be detected at a wide solid angle.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a nucleic acid base sequencing device, and particularly to an electrophoresis gel suitable for detecting nucleic acid fragments with high sensitivity.

[Conventional technology]

Conventionally, base sequencing of nucleic acids has been determined using methods such as the Maxam-Gilbert method (Method in Engineering).
ds in Engymology) 65. pp
, 560-581). in this way.

First, a nucleic acid labeled with a radioactive isotope is chemically fragmented, and then the nucleic acid fragments of different lengths are aligned in a molecular baby face in a gel support sandwiched between glass plates using electrophoresis. The gel support is then peeled off from the glass plate and an autoradiogram is taken to detect the migration band containing radioactive nucleic acid fragments and determine the base sequence of the nucleic acid. Here, a conventional method for determining the base sequence of a nucleic acid fragment will be explained based on one drawing. FIG. 3 is a perspective view showing the structure of a conventional electrophoresis device for nucleic acid fragments. As shown in the figure,
Gel for electrophoretic separation of nucleic acid fragments sandwiched between two glass plates 3 2. It is composed of an electrode liquid tank 1 in which both ends of the electrophoretic separation gel 2 are immersed, and a DC power source 6. and radioactive isotopes (
For example, a nucleic acid fragment sample labeled with 32P) was supplied to the negative electrode side slot 5 of the electrophoretic separation gel 2, and the length of the gel was adjusted. !
1) When electrophoresed at a voltage Ev of about 40 V/port, nucleic acid fragments having the same molecular weight migrate from the negative electrode toward the positive electrode, forming migration bands 4, with a mobility approximately inversely proportional to the logarithm of one molecular weight. From the migration pattern of this nucleic acid fragment in the migration zone 4, the entire sequence of the nucleic acid base molecule is determined.

At this time, in order to read the pattern, the method generally used is to take an autoradiogram as described above. Autoradiography has a problem in that it takes a long time (50 hours or more) to transfer the X-ray film because the amount of nucleic acid labeled with a radioactive isotope is small.

Furthermore, in order to compensate for the vertices during the above-mentioned autoradiography, a method of directly detecting β rays from nucleic acid fragments is being considered to speed up the process. That is, a cross-sectional view of the electrophoresis device is shown in FIG. 4, and by detecting β rays when the nucleic acid fragments 4 being electrophoresed pass through the detector 30, autoradiography is not necessary and high speed is possible. This is an attempt to make the world a better place.

Next, a cross-sectional view of the detector 30 is shown in FIG. The electrophoretic separation gel 2 and the nucleic acid fragments 4 to be electrophoresed pass through the slits 10 one after another. The β rays 7 from the nucleic acid fragment 4 pass through the slit for 10 minutes and enter the scintillator 11.

The β rays 7 are converted into fluorescence 8 without losing energy by the scintillator 11. This fluorescence 8 is converted into a current by a secondary electron multiplier phototube 12 and coupled to a signal processing circuit 13 . As a result, as shown in FIG. 5, the output of the signal processing circuit 13 is obtained as a waveform representing the number of counts of β-rays with respect to the passage time of the detector. Nucleic acid fragments 4 can be measured by processing this waveform by computer 14.

[Problem that the invention seeks to solve]

The conventional technology for directly detecting β-rays does not necessarily give sufficient consideration to detection sensitivity, and there are problems with the accuracy of base sequence determination.

An object of the present invention is to provide a highly accurate sequencing device by directly detecting nucleic acid fragments with high sensitivity.

[Means for solving problems]

The above object is achieved by mixing particulate or liquid scintillator with a part of a separation gel for electrophoresis and detecting fluorescence from this scintillator.

Although the scintillator may be included throughout the electrophoretic separation gel, it is preferable that the scintillator be included only in a predetermined portion, as will be clear from the effects described below.

[Effect]

The scintillator mixed into the electrophoresis separation gel has a diameter of 100 μm.
It is preferable to use the following granular form or liquid form mixed with gel. When the electrophoresed nucleic acid fragments pass, the particulate or liquid scintillator emits fluorescence due to β rays from the radioisotope labeled on the nucleic acid fragments.

This fluorescence is converted into electric current by a phototube and coupled to a signal processing circuit.

As a result, nucleic acid fragments migrate between granular or liquid scintillators, and the emitted β rays can illuminate the scintillator over the entire solid angle, making it possible to convert most of the β rays into fluorescence. .

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. Components having the same configuration as the conventional example are given the same reference numerals. The particulate scintillator 20 according to the present invention is mixed into the detection portion of the electrophoretic separation gel 2. In addition, the reflecting mirror 21 is a secondary electron multiplier phototube (
PMT) 12.

As a result, the nucleic acid fragments 4 that migrate are transferred to the particulate scintillator 20.
When passing through the contaminated area of the particle scintillator 20, the β rays 7 from the radioisotope that labels the nucleic acid fragment pass through the particulate scintillator 20.
It is converted into fluorescent light 8.

Further, the fluorescent light 8 is efficiently collected on the PMT 12 by the reflecting mirror 21.

Usually, the thickness of the gel is about 500 μm, but in this case, the diameter of the granular scintillator was 100 μm or less. The material of the scintillator may be a known plastic scintillator. Since the energy loss of β-rays to this scintillator is 200 K e v / mz, the energy loss of β-rays in one granular scintillator is 20Key
/100μm.

The average energy of radioisotope 32p is IMe
v, one granular scintillator provides an intensity of 2% of the average fluorescence intensity. Also, the thickness of the β-ray is 50
When emitted from the center of a 0 μm nucleic acid fragment, the β-rays are 3 particulate scintillators? Since the light passes through the light, an average fluorescence intensity of 6 channels can be obtained.

By the way, as shown in FIG. 1, the conventional scintillator 11
By combining this with the present invention, β-rays from the nucleic acid fragment 4 can be detected over almost any solid angle. Further, in this case, by coating the side surface of the scintillator 11 with a reflective material 22, the fluorescent light 8 can be effectively focused onto the PMT 12.

Further, if the reflecting mirror 21 shown in FIG. 1 comes into direct contact with the gel 2, it will be contaminated with radioisotopes.

For example, it is preferable to cover with a film 23 that is easy to wash. Furthermore, since the detection system shown in FIG. 1 detects weak fluorescence, it is necessary to store the electrophoretic separation gel and the detector in a dark box.

Furthermore, one embodiment of the present invention is shown in FIG. The configuration is such that the conventional scintillator 11 and slit 10 are omitted. This makes it possible to detect nucleic acid fragments with a simpler configuration than in the past, making it a much more practical device.

Although particulate plastic scintillators were used in the embodiments shown in FIGS. 1 and 2, similar effects can be obtained by mixing an organic liquid scintillator with the electrophoretic separation gel in the present invention. Since these photosensitized electrophoresis gels are used as single-use materials as in the past, low cost is one of the criteria for selecting materials for scintillators.

〔Effect of the invention〕

According to the present invention, as described above in detail.

Since β-rays from nucleic acid fragments can be measured over a wide solid angle, the reliability of analysis is greatly improved. In particular, when only a small amount of nucleic acid fragments are obtained, the amount of β-rays to be detected will be small; however, according to the present invention, highly reliable measurement is possible even for a small amount of fragments.

[Brief explanation of the drawing]

Figures 1 and 2 are explanatory diagrams explaining the relationship between the photosensitized electrophoresis gel and the detector according to the present invention, and Figures 3, 4, and 5 are explanatory diagrams of the conventional method for determining the base sequence of nucleic acids. It is a diagram. 1... Electrolytic solution tank, 2... Electrophoretic separation gel, 3... Glass plate. 4... Nucleic acid fragment, 5... Slot, 6... DC power supply, 7... Beta ray, 8... Fluorescence, 10... Slit, 11... Scintillator, 12... Phototube , 21・
...Reflector, 22...y] 1 Mouth': f12 Fig. 3 Turning force 4 Mouth Fig. 5

Claims (1)

[Claims] 1. An electrophoresis gel for electrophoretically separating nucleic acid fragments labeled with a radioisotope, wherein the electrophoresis gel contains a scintillator capable of converting radiation from the radioisotope into light. Electrophoresis gel.