JPH01112146A - Base sequencing device - Google Patents

Abstract

PURPOSE:To obtain a large quantity of emitted light by projecting excitation light completely independently to each migration lane and casting the excitation light along the longitudinal direction of the bands where the nucleic acid fragments appearing in the respective lanes are separated. CONSTITUTION:The primers of the DNA fragments are previously labeled with FITC by a Sanger method and are loaded as samples into the respectively separate migration cells 2 by each kind of the terminal bases. The migration is then started by correctly disposing respective electrolyte cells and starting energization. The excitation light beams are projected through light guides 3 into the gels simultaneously with the migration. Fluorescence is emitted when the bands separated of the DNA fragments arrive at the beams of the excitation light on progression of the migration. The fluorescence is picked up by optical fiber bundles 4 and is guided to a detector part 6. The light signals of the fluorescence obtd. in such a manner are subjected to A/D conversion and the converted signals are subjected to data processing in a signal processing part, by which the base sequence of the DNA is determined.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to the step of determining the base sequence of a nucleic acid by Sanger's method, in which the primer is labeled in advance with a labeling dye such as a fluorescent substance or a phosphorescent substance. Ok, the final stage) V
This invention relates to an apparatus for reading sequences from electrophoresis using a spectroscopic method using light emitted from a labeled dye.

(Prior Art) There are two possible fluorescent reading methods for bands of nucleic acid fragments developed by Ge/I/electrophoresis: an online method and an offline method.

In the online method, time changes in fluorescence at a point on the migration lane are read while electrophoresing nucleic acid fragments in a migration gel; in the offline method, a separately run migration gel is attached to a dedicated reading device after the migration is complete. and read the migration pattern.

Sanger method (Proc, Natl, A, cad, 8c
i, USAJ magazine.

Vol. 74, p. 5463 (1977)), the terminal base of each sample is A (adenine).

One set of samples consists of four types of nucleic acid fragment samples that are either G (guanine), T (thymine), or C (cytosine). One type of sample with one of four types of terminal bases is
If you try to run multiple samples simultaneously in one lane, the off-line method requires fluorescence measurement in two-dimensional directions of the slab-like migration gel, and even the online method requires sample alignment on the migration gel. One-dimensional high-speed fluorescence measurements in the direction are required.

All of the slab-type electrophoretic gel fluorescence measuring devices that have been announced so far are online methods.

The easiest way to realize fluorescence measurement is to use the apparatus shown in Figure 6 (r HighTechn
The device described in Science J magazine, December 1986 issue, page 49 also falls into this category.

In FIG. 6, reference numeral 51 represents a migration gel made of polyacrylamide, and both ends of the gel are immersed in birch tanks 52 and 53. The electrode tanks 52 and 53 contain an electrolytic solution. A migration voltage is applied between the electrode tanks 52 and 53 by a migration power source 54. A slot 55 for injecting a sample is provided at one end of the electrophoresis gel/l 151. A sample with terminal base printing is injected into this slot 55, and the sample is transferred to the electrophoresis gel/l 151 by the electrophoresis voltage from the electrophoresis power supply 54.
It is electrophoresed inside in the direction of arrow 56 and is completely expanded.

Reference numeral 57 denotes a laser serving as an excitation light source; the excitation light is reflected by a half mirror or dichroic mirror 58, and is irradiated onto the electrophoretic gel 51 through an objective lens 59. Fluorescent light from the fluorescent label of the sample that has migrated through the electrophoresis gel 1151 is again focused by the objective lens 59.

The light passes through a half mirror or dichroic mirror 58, passes through a fluorescence selection interference filter 6o, and is received and detected by a photomultiplier tube 61 as a photogravitational element.

In the apparatus shown in FIG. 6, one objective lens 59 is used for both excitation light irradiation and fluorescent light reception, and the objective lens 59 and the entire optical system associated with it are arranged in the sample arrangement direction 62 (migration direction 5).
The scanning is performed mechanically in a direction perpendicular to 6 (transverse direction in FIG. 9).

Another device for fluorescence measurement of slab-like migration gels is shown in Figure 7 (Proceedings of the Biophysical Society of Japan, 3E1).
130 (October 1986)).

The excitation light from the laser 57 as the excitation light source is collected from the end face of the electrophoresis gel/L' 51 by the condenser lens 63.
51 in a direction parallel to it. The fluorescent light is received one-dimensionally or two-dimensionally at once by a fluorescent light receiving lens 64 in the normal direction of the surface of the electrophoretic gel 51, and is then passed through a fluorescent light selection interference filter 6.
0 and is amplified by the image intensifier 65, -
A dimensional or two-dimensional photodetector 66 receives and detects the light.

(Problems to be Solved by the Invention) In the base sequencing apparatus shown in Figure @6, fluorescence is measured in the direction of reflection of the excitation light, so the background light with strong Y-scattering of the excitation light cannot be used. Therefore, the S/N ratio deteriorates. Rayleigh scattering is strong in the forward and backward directions, and weak in the 90 degree direction.

In addition, in the apparatus shown in FIG. 6, the objective lens 59 and the like.

All or part of the excitation optical system and the receiving optical system must be mechanically scanned. Online measurement requires scanning all lanes in a sufficiently short time compared to the migration speed, but such precision optical systems are generally heavy and have large inertia.

Such high speed scanning is either not possible at all or would be very expensive.

In the apparatus shown in FIG. 7, the electrophoretic gel 51 is extremely large compared to the diameter of the fluorescent light receiving lens 64 that is normally available. To compensate for this, the detection section must be one-dimensional or two-dimensional, and the amplification must be increased. For example, an image intensifier or the like must be used for this purpose, but such elements are very expensive.

In addition, if the electrophoresis gel is thin, the laser beam may not be narrowed down and may protrude from the electrophoresis gap, and if the electrophoresis gel is not exactly straight and bends at the point where the excitation light is incident, there are problems such as measurement failure. be.

Furthermore, if two DNA bands simultaneously appear on the laser beam passing through the gel in different lanes due to electrophoresis, there will be some difference between the DNA band that passes through the beam first and the one that passes later. This may be due to a quantitative discrepancy in the fluorescent light.

In the present invention, the excitation light beam for each migration lane is passed independently through the thickness of the migration gel.

The purpose is to equalize the conditions for generating fluorescent light and eliminate the differences that occur between lanes.

(Means for Solving the Problems) The present invention comprises an electrophoresis cell section in which electrophoresis lanes for electrophoresing nucleic acid fragments labeled with a labeling dye are separated for each type of terminal base of the nucleic acid fragment, and A mechanism that allows excitation light to enter within the range of the rubbing direction of the gel, and receives light emitted from the labeled dye of the nucleic acid fragment developed in the electrophoresis cell from a direction perpendicular to the direction of the excitation light in each electrophoresis cell section. It consists of a light receiving mechanism.

Note that the S configuration in which the excitation light is incident is configured to sequentially scan at high speed using a light guide or a lens system for beam convergence.

(Function) The present invention allows excitation light to be incident on each migration lane completely independently, and moreover, allows nuclei to appear in each lane.

Excitation light is applied along the ``longitudinal direction'' of the separated bands of acid fragments to obtain a greater amount of light emission.

(Example) An example of the present invention will be described based on the drawings. Figure 1 shows
It is a diagram mainly showing the electrophoresis cell part. 1 is the electrophoresis center /I/
2 and the electrophoresis tank 5 on the negative side are attached and fixed, and at the same time, it also serves as the anode side electrode liquid tank. The electrophoresis center/L' of 2 is.

Figure 4 shows the configuration of a single part of the electrophoresis cell, which is installed in four cells to allow completely independent electrophoresis for each type of terminal base (A, G, C, T) of a nucleic acid fragment. .

FIG. 4 (al is a plan view, FIG. 4 (bl is a front view, and FIG. 4 (C) is a right side view. 22.23 is a support plate,
They are arranged parallel to each other at intervals, and a polyacrylic/reamide migration gel 24 is formed in the space.

As the support plates 22 and 23, glass plates or acrylic/I/resin plates can be used, and when ultraviolet light is used as excitation light, quartz glass plates are used. Inside the electrophoresis cell 2,
A light guide 3 is provided to allow excitation light to enter within a range in the thickness direction of the gel. Also.

An optical fiber bundle 4 is attached to the cell/I/2 in order to extract the luminescence emitted by the labeled dye of the nucleic acid fragment developed in the electrophoresis gel from a direction perpendicular to the direction of the excitation light. The light emitted from the optical fiber bundle 4 is guided to a detector section 6 including an interference field for fluorescence selection and a photomultiplier tube.

As an example of the optical fiber bundle 4, for example, ESKA (product of Mitsubishi Rayon Co., Ltd.) can be used. The detection signal from the detector section 6 is converted into a digital signal and processed by the signal processing section.

FIG. 2 shows means for making excitation light enter the light guide 3 provided in each electrophoresis cell/l/2 one after another at high speed.

Reference numeral 7 denotes an argon laser as an excitation light source that generates an excitation light beam, and 9 denotes a reciprocating rotating mirror that scans the excitation light beam, which is the laser light from the argon laser 7, in the direction of the electrophoresis cell 2. Argon laser is 488nm and 514n
It emits light of m.

By the way, for example, FIT is used as a fluorescent labeling dye to label a sample.
When using C'i, the fluorescence spectrum) /l/ peak is 5
Since it is around 20 nm, the wavelength of the A7Regon laser is 51
Light due to 4 nm oscillation becomes an interference.

In addition, since the excitation wavelength peak of this fluorescent labeling dye is close to 488 nm,
An excitation light selection interference filter 16 (a filter with a sufficiently narrow half-width) having a transmission center wavelength of 488 nm is inserted immediately after the argon laser 7. Interference filter 1 for excitation light selection
A condensing lens 8 is installed between the rotating mirror 6 and the rotating mirror 9 for converging the excitation light beam into a minute spot inside the electrophoretic gel to improve resolution.

A cylindrical lens 10 is installed to provide a converging function in the scanning direction of the excitation light beam over the range scanned by the excitation light beam, and a cylindrical lens 11 is installed to prevent unnecessary disturbance light from entering. Contains 10 full lenses and 3 light guides IIIK.

The operation of this embodiment having the above configuration will be described below.

As a sample, a primer for a DNA fragment was pre-coupled with FITC (520r1m when excited at 488nm) using the Sanger method.
Label with a nearby fluorescent substance) and separate two electrophoresis cells for each type of terminal base (4 types: A, G, C, and T).
Load against. Then, each electrode solution tank is placed correctly and electricity is turned on to start electrophoresis. Electrophoresis center during electrophoresis
Figure 5 shows state a.

Simultaneously with electrophoresis, an excitation light beam is made to enter the inside of the gel through the light guide 3. When the electrophoresis progresses and the separated bands of the DNA fragments reach the excitation light beam, fluorescent light is emitted, which is collected by the optical fiber bundle 4 and guided to the detector section 6.

The fluorescent light signal thus obtained is ~■ converted.

The signal processing section processes the data and determines the base sequence of the DNA.

It is also conceivable that instead of the light guide 3, a suitable focusing lens system 1, for example as shown in FIG. 3, may be coupled to the electrophoresis center 2. Qualified convergence lens ih, convergence optical fiber 12. Tanrem lens 13° aperture 14.
It consists of a converging lens 15. in this case.

The tip of the optical fiber 12 extending toward the laser light source is.

They are arranged on a circumference of an appropriate radius centered on the rotation axis of the rotating mirror 9, with central angles at equal intervals. If this method is 4
It is also possible to have a large number of lanes.

Furthermore, a light guide may be provided at a location where the excitation light beam that has completed passing through the gel exits to the opposite side to reduce scattering that occurs at the interface with the electrophoresis cell 2.

(Effect) According to the present invention, since the excitation light is applied to the "longitudinal direction" of the separated bands of DNA fragments appearing in each lane, the amount of fluorescent light emitted from one band (absolute amount) increases. You can expect it.

The excitation light beam is incident on each lane completely independently, so if the beam is incident on one flat gel from its edge, the number of lanes increases and the beam transmission distance becomes There is no difference in the state of excitation between the first and last lanes, resulting in a uniform condition.

Furthermore, since the distance of the excitation light beam passing through the gel becomes shorter, the parallelism between the interface between the gel and the electrophoresis cell and the incident beam can be easily adjusted. Further, a considerable amount of scattered light components at the interface between the gel 7 and the electrophoresis cell can be canceled.

[Brief explanation of the drawing]

Figure 1 is a diagram mainly showing the electrophoresis cell section of the present invention;
The figure mainly shows the method of incidence of excitation light, Figure 3 shows a suitable converging lens system, and Figure 4 shows (a) to 4.
(Figure 5 showing the shapes of individual parts of the C1 migration cell.
The figure is a state diagram during electrophoresis, Figure 6. FIG. 7 is a conventional diagram. 2-Electrophoresis center/I/3...Light guide 4...Optical fiber bundle 7-.Laser 10-...Cylindrical lens 16-.Interference filter [;・)Ilf-J::

Claims (1)

[Claims] 1. An electrophoresis cell section in which the electrophoresis lane for electrophoresing nucleic acid fragments labeled with a labeling dye is separated for each type of terminal base of the nucleic acid fragment, and excitation light is incident within the range in the thickness direction of the gel within the cell section. and a light receiving mechanism that receives light emitted from a labeling dye of a nucleic acid fragment developed in an electrophoresis gel in a direction perpendicular to the direction of excitation light in each electrophoresis cell section.