JP3127544B2 - Base sequence determination method and apparatus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for determining a base sequence of DNA or the like.

[0002]

2. Description of the Related Art Conventionally, the base sequence of DNA or the like has been determined by, for example, the Maxam-Gilbert method. In this method, first, a DNA fragment labeled with a radioisotope is chemically fragmented, then arranged in order of molecular weight by electrophoresis, and detected by autoradiography.

[0003] However, although this method is excellent in detection sensitivity, there is a problem in safety due to the use of radioisotopes. At present, a method of labeling with a fluorescent substance instead of a radioisotope has been proposed (eg, , JP-A-60-242368
, Nature, Vol. 321, No. 12, pp. 674-679 (1986)).

[0004]

In the method using a fluorescent substance, the electrophoretic DN, which is the last step of sequencing, is used.
There was a problem in detecting the A fragment. In other words, in this method, the fluorescent labeling substance must be irradiated with excitation light and its fluorescence must be detected. Raman scattering due to the water in the water sometimes interfered with the fluorescent light. Therefore, at present, the fluorescence method is inferior in sensitivity by one to two orders of magnitude compared to the method using a radioisotope.

In order to solve this problem, a method has been proposed in which the background is reduced by irradiating excitation light in a pulsed manner and detecting fluorescence only after the excitation light is quenched. Since the emission time of the fluorescent light is very short, it is necessary to switch the fall time of the excitation light pulse to nanoseconds or less, and the feasibility is poor.

Accordingly, the present invention has been made to solve the above-mentioned problems at once by using a DNA without using a radioisotope or a fluorescent substance.
An object of the present invention is to provide a method for labeling a fragment and determining a nucleotide sequence.

[0007]

According to the present invention, in order to solve the above problems, a DNA fragment whose base sequence is to be determined is cut into a plurality of DNA fragments, and the cut DNA fragment is labeled with an enzyme or a substrate and then subjected to electrolysis. Electrophoresis and detecting the labeled DNA fragment.

Here, the DNA fragment whose base sequence is to be determined is not particularly limited, and DNA fragments extracted from human biological fluids
Anything is fine. DNA extraction is performed by a known method such as solvent extraction using an organic solvent and centrifugation.

The cutting of the DNA fragment is performed by restriction enzyme treatment. Restriction enzymes are DNA degrading enzymes (deoxyribonucleases) that recognize and cut a specific base sequence of DNA.
Thus, a long DNA can be neatly cut into various fragments to obtain a specific DNA fragment. Examples of the type of restriction enzyme include, but are not limited to, EcoRI, PstI, and SalPI.

[0010] One end (5 'end or 3
The 'end' is labeled with an enzyme or a substrate, and which one to label depends on the type of detection system. That is, when an enzyme is added to the detection system to carry out the enzyme reaction, the substrate is labeled, and when the enzyme reaction is reversed, the enzyme is labeled. Examples of the combination of the substrate and the enzyme include glucose / glucose oxidase, L
-Amino acid / L-amino acid oxidase, L-arginine / arginine decarboxylase, urea / urease, neutral lipid / lipase, 4-methylumbelliferyl phosphate / alkaline phosphatase, parahydroxyphenylpropionate / peroxidase and the like. However, the present invention is not limited to these. Cleavage substrate DN
To label one end of the A fragment, it is bound to the DNA via a substance having good binding properties. The substrate is preferably dispersed with a plasticizer or the like.

When labeling an enzyme, a known method (for example, Murakami, A. et al, Nucl. Acid Res. 1989.17.5587-559)
Perform according to 5). The type of the substrate and the enzyme used for labeling may be one type or may be other types depending on the type of base (A, G, T, C).

After labeling the DNA, it is subjected to ordinary electrophoresis. As the electrophoresis, known gel electrophoresis can be used, and a polyacrylamide gel is generally used as the gel.

When the substrate is labeled, the detecting means is preferably performed with an enzyme electrode. The enzyme electrode is configured by combining an enzyme-immobilized membrane and an electrode.For example, when labeling glucose, a glucose sensor manufactured by combining a glucose oxidase-immobilized polyacrylamide gel membrane and a Clark-type oxygen electrode is used. . The arrangement of the enzyme electrodes is performed for each migration lane of the migration plate.

When the enzyme is labeled, a substrate solution is added to the whole electrophoresis plate after the electrophoresis, and an enzyme reaction is carried out for detection. The result of the enzymatic reaction can be performed, for example, by irradiating the electrophoresis plate with light and measuring the absorbance, but is not limited thereto.

The determination of the base sequence based on the detection signals of the labeling substrate and the enzyme can be performed by a known method.

[0016]

According to the present invention, since the base sequence can be determined by utilizing an enzyme reaction, safety problems such as when using radioisotopes and complicated optical systems when using fluorescent substances are not required. .

[0017]

FIG. 1 shows an embodiment of an apparatus for carrying out the method of the present invention. In FIG. 1, reference numeral 2 denotes an electrophoresis plate, which is constituted by holding a slab gel of polyacrylamide between glass plates. One end of the electrophoresis plate 2 is affected by the electrolyte in the electrode tank 4, and the other end is affected by the electrolyte in the electrode tank 6. The electrolyte in the electrode tank 4 and the electrolyte in the electrode tank 6 are electrically connected to a power supply 10 for electrophoresis via electrodes (not shown).

At one end of the electrophoresis plate 2, a plurality of electrophoresis slots 8 for injecting a sample are formed. In the figure, A is adenine at the terminal base, G is guanine, T is thymine,
C means a slot for injecting a sample that is cytosine, and one set of four types is used. When the electrophoresis lanes are divided according to the types of terminal bases, only one type of labeling substance may be used.

In the electrophoresis direction of the electrophoresis plate 2 (X direction in the figure),
An enzyme electrode section 9 is provided. The enzyme electrode section 9 includes a glass electrode holding plate 1 and an electrode tube 3, and the electrode tube 3 accommodates an enzyme electrode. The number of the electrode cylinders 3 corresponds to the number of migration slots, that is, the number of migration lanes on a 1: 1 basis. The tip of the electrode in the electrode tube 3 contacts the gel of the electrophoresis plate 2.
The signal from the electrode is taken into the signal processing unit 5 and sent to the analysis unit 7.

The determination of the base sequence of a sample with the above configuration is performed as follows. First, the 5′-end or 3′-end of a DNA fragment whose ends have been aligned by restriction enzyme treatment is labeled with an enzyme substrate. After labeling, base chemical modification is performed. This method can be performed by the well-known Maxam-Gilbert method,
After this reaction, a treatment such as ethanol precipitation is performed to obtain a base-specific cut DNA strand.

The thus obtained DNA fragment is placed in the electrophoresis slot 8 corresponding to each type of terminal base.
The DNA fragment sample placed in the electrophoresis slot 8 is electrophoresed in the X direction by applying an electrophoresis voltage between the electrode bath 4 and the electrode bath 6 by the electrophoresis power supply 10.

Since an enzyme electrode is provided in the electrophoresis direction, the presence of the labeled substrate can be detected by the enzyme electrode, and the signal is sent to the signal processing unit 5 and the analysis unit 7 to determine the base sequence. .

[0023]

According to the present invention, safety problems such as when using a radioisotope as a labeling substance, and a complicated optical system when using a fluorescent substance become unnecessary.

[Brief description of the drawings]

FIG. 1 shows an apparatus for implementing the method of the invention.

[Explanation of symbols]

 2: Electrophoresis plate 4, 6: Electrode tank 9: Enzyme electrode part

Claims (2)

(57) [Claims] 1. A method comprising the steps of: cutting a DNA fragment whose base sequence is to be determined into a plurality of DNA fragments; labeling the cut DNA fragment with an enzyme or a substrate; and performing electrophoresis to detect the labeled DNA fragment. Nucleotide sequencing method. 2. An injection slot into which a cut DNA fragment labeled with a substrate is injected, an electrophoresis plate for electrophoresing the injected cut DNA fragment, an enzyme electrode provided for each electrophoresis lane of the electrophoresis plate, A base sequence determination device comprising: an analysis unit that determines a base sequence based on a signal.