JPS63222264A - Determination of base sequence of nucleic acid by chemiluminescence detection - Google Patents

Abstract

PURPOSE:To achieve a measurement with an inexpensive device and with a high S/N ratio, by a method wherein a nucleic acid fragment labeled by a chemiluminescent substrate is eluted by electrophoresis, a reagent for emission of light is made to react with an elution sample to detect chemiluminescence optically. CONSTITUTION:A DNA fragment labeled by N-(4-aminobutyl)-N-ethylisoluminol or the like is injected at one end of migration columns 2-1-2-4 and an electrophoresis is performed by a phoretic voltage from a power source 18. Bands (a), (b)... of the DNA fragment eluted from the other end of the migration columns 2-1-2-4 are carried through a passage 10 by a buffer 14. Then, hydrogen peroxide, potassium ferricyanide and caustic soda are mixed sequentially and then, a solution is introduced into a flowcell 16 to detect chemiluminescence. The results of detection are processed by a signal processing section 34 to determine the sequence of DNA.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention electrophores nucleic acid fragments such as DNA labeled with a labeling substance, and optically detects the a-textured material after electrophoresis, thereby detecting the nucleic acid bases. It relates to a method for determining sequences.

(Prior art) Conventional methods for determining DNA base sequences using dye labeling methods include fluorescent dye methods that use fluorescent substances as labeling dyes (for example, Japanese Patent Application Laid-Open No. 60-2208
(See Japanese Patent Laid-Open No. 60-242368, etc.).

In the fluorescent dye method, Rayleigh scattering of excitation light by the gel and Raman scattering (Stokes lines) by water interfere with a small amount of fluorescent light, increasing the background.

It is said that the constant fluorescence method, in which excitation light is continuously irradiated, is one to two orders of magnitude less sensitive than the radioisotope method. Therefore, the error rate has reached more than 1% (rNatur
(Refer to reJ, Vol. 321, No. 12, pp. 674-679).

Therefore, in the fluorescent dye method, a time-resolved fluorescence method has been proposed in place of the steady fluorescence method (see Japanese Patent Laid-Open No. 61-2077). In the time-resolved fluorescence method, the background is reduced by irradiating excitation light in a pulsed manner and detecting the fluorescence only after the excitation light is quenched (for example, "Fluorescence Measurement - Application to Biological Sciences" (Society Publishing) Center, 19
(published in 1983), see pages 99-160).

(Problems to be Solved by the Invention) In the time-resolved fluorescence method, since the emission time of fluorescence is very short, switching is required to make the fall time of the excitation light pulse 1 nanosecond or less. Such high-speed switching cannot be achieved with a mechanical shutter and requires the use of a mode-locked laser. Therefore, the device becomes expensive. In addition, high-speed signal processing is difficult, and the time-resolved fluorescence method is currently only a proposal.

Furthermore, gel supports used in electrophoresis devices are made of glass or polyethylene, and some of these supports exhibit weak fluorescence. Therefore, even if a time-resolved fluorescence method was adopted, it would not be possible to analyze DNA by itself.
There is no guarantee that fragments can be detected with sufficiently high sensitivity.

In place of the fluorescence method in which a fluorescent label is excited by an excitation light source, the present invention uses a chemiluminescent substrate to label the nucleic acid fragments, performs a chemical reaction after eluting the nucleic acid fragments from an electrophoresis gel, and optically detects the nucleic acid fragments. The object of the present invention is to provide a base sequencing method that does not require an expensive excitation light source such as a laser, is inexpensive, and can increase the S/N ratio.

(Means for Solving the Problems) In the present invention, one end of the gel of an electrophoresis device is connected to a flow path, and the other end of the gel is provided with a terminal base species of a nucleic acid fragment labeled with a chemiluminescent substrate. A reaction reagent that reacts with the chemiluminescent substrate to emit light is supplied to the channel, and the channel is guided to a flow cell to elute the chemiluminescence from one end of the gel through electrophoresis. Detect and determine the base sequence.

(Example) The figure shows an example of a base sequence determination device used in the present invention.

2-1 to 2-4 are electrophoresis columns in which polyacrylamide gel is filled in a glass tube having an inner diameter of about 1 mm, for example. The samples include four types of D adjusted to the type of terminal base.
Since NA fragments are used, a set of four electrophoresis tubes is used. One end of each of the electrophoresis columns 2-1 to 2-4 is immersed in a buffer solution 6 placed in a one-sided electrode tank 4, and a DNA fragment sample is injected from each end.

The other end of each of the electrophoresis columns 2-1 to 2-4 is connected to the channel 10 by a mixing connector 8. The buffer solution 14 in the + side electrode tank 12 is supplied to the flow path 10 by a pump 15, flows through the flow path 10, and is guided to the flow cell 16.

A power source 18 for electrophoresis is provided between the one side electrode tank 4 and the + side electrode tank 12.
is provided, and a migration voltage is applied to the migration columns 2-1 to 2-4.

A mixing connector 20 connects hydrogen peroxide (H2
0=) A flow path for water is connected, and the hydrogen peroxide aqueous solution tank 22
A hydrogen peroxide solution is supplied to the channel 10 by a pump 24 from the pump 24 .

A flow path for a potassium ferricyanide (K3 Fe (CN)s) aqueous solution is further connected to the flow path between the mixing connector 8 and the flow cell 16 by a mixing connector 26, and a flow path for potassium ferricyanide (K3 Fe (CN)s) aqueous solution is connected to the flow path between the mixing connector 8 and flow cell 16.
An aqueous solution of potassium ferricyanide and caustic soda is supplied to the channel 10 by a pump 30 from a tank 28 of an aqueous solution containing NaOH).

A photomultiplier tube 32 is provided close to the flow cell 16, and the light emitted from chemiluminescence in the flow cell 16 is detected by the photomultiplier tube 32 and guided to a signal processing section 34 using a microcomputer to determine the base sequence of the DNA. is determined.

The DNA fragment sample injected into one end of the electrophoresis column is a group of DNA fragments obtained by labeling an oligonucleotide primer with an aminated end with a chemiluminescent substrate and performing a chain elongation reaction using the Sanger method. N-(4-aminobutyl)-N-ethylisoluminol was used as the chemiluminescent substrate, and the process of labeling with this chemiluminescent substrate was described in "J.

of ChromatographyJ magazine, No. 328,
This is carried out according to the method for labeling amines described on pages 121 to 126 (1985).

DNA fragments labeled with N-(4-aminobutyl)-N-ethylisoluminol with different terminal bases A (adenine), G (guanine), T (thymine) and C (cytosine) were added to each electrophoresis column 2- 1 to 2-4, and electrophoresis is performed using the electrophoresis voltage from the electrophoresis power source 18.

Although only the configuration related to the flow path 10 provided at the other end of the electrophoresis column 2-1 is shown in the figure, other electrophoresis columns 2-2
A flow path 10 having the same configuration is also connected to the other end of ~2-4, and each flow path 10 is guided to a respective flow cell 16 so that chemiluminescence is detected.

When a DNA fragment sample is injected into one end of each electrophoresis column 2-1 to 2-4 and electrophoresis is started at the same time, each electrophoresis column 2
Bands a, b, etc. of the migrated DNA fragments are sequentially eluted from the other ends of -1 to 2-4 by the migration buffer 14 of the secondary electrode tank 12, and the flow path 10 is transferred to the flow cell 16. being carried in the direction.

Chemiluminescence is produced by sequentially mixing appropriate concentrations of hydrogen peroxide, potassium ferricyanide, and caustic soda into a solution containing this labeled DNA fragment. The solution that has started chemiluminescence is immediately introduced into the flow cell 16 and detected by the photomultiplier tube 32 by photon counting. Repeat this operation for sample A of each electrophoresis column 2-1 to 2-4.
,G.

The signal processing unit 34 performs the processing for the four types of T and C.
to determine the DNA base sequence.

The mechanism by which N-(4-aminobutyl)-N-ethylisoluminol reacts with hydrogen peroxide and potassium ferricyanide to emit chemiluminescence is described in the above-mentioned document rJ, of Chro+
It is described in the magazine MatographyJ.

Reaction systems that produce chemiluminescence are not limited to the system of isoluminol derivatives and potassium ferricyanide. for example,
By using a compound that reacts with hydrogen peroxide to generate a molecular species (such as dioxetanedione) that chemically excites a fluorescent substance, DNA fragments labeled with a fluorescent dye can be chemiluminescent without being excited by a light source. can be detected.

Regarding the mechanism of chemically exciting fluorescent substances, HLPC
An example in the analysis is rJ, of Liquid Chro.
MatographyJ Magazine, Volume 6, No. 9, No. 160
3-1616 pages (1983).

In this case, a fluorescent substance is used as the chemiluminescent substrate, and the DNA fragments are labeled with the fluorescent substance as is done in conventional fluorescence methods. Instead of the aqueous solution tank 28 of potassium ferricyanide and caustic soda, an aqueous solution of a compound that reacts with hydrogen peroxide to produce excited molecules of a fluorescent substance such as dioxetanedione may be supplied.

(Effects of the Invention) In the present invention, a nucleic acid fragment labeled with a chemiluminescent substrate is electrophoresed and eluted, and the eluted sample is reacted with a reaction reagent that reacts with the chemiluminescent substrate to emit light, and is guided to a flow cell. Since the base sequence is determined by optically detecting chemiluminescence, compared to conventional fluorescence methods, it does not require an expensive excitation light source such as a laser, making the device less expensive.

Furthermore, since no light excitation is performed, background such as stray light can be kept low. Therefore, the S/N ratio is improved.

Furthermore, since a complicated optical system such as lenses and mirrors is not required, adjustment is easy.

[Brief explanation of drawings]

The figure is a schematic diagram showing one embodiment. 2-1 to 2-4...Migration column, IO...
...Flow path. 16...Flocel, 22...Hydrogen peroxide aqueous solution tank, 28...
- Potassium ferricyanide and caustic soda aqueous solution tank, 32... photomultiplier tube, 34... signal processing section.

Claims (2)

[Claims] (1) One end of the gel of an electrophoresis device is connected to a flow channel, and nucleic acid fragments labeled with a chemiluminescent substrate are injected into the other end of the gel for each terminal base type, and electrophoresed to form one end of the gel. A method in which a reaction reagent that reacts with the chemiluminescent substrate to emit light is supplied to the channel, and the channel is led to a flow cell to optically detect chemiluminescence to determine the base sequence. (2) N-(4-aminobutyl) as chemiluminescent substrate
2. The method according to claim 1, using -N-ethylisoluminol and using hydrogen peroxide and potassium ferricyanide as reaction reagents.