JPH0734005B2 - Nucleic acid nucleotide sequencer - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for determining a base sequence in a nucleic acid such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).

[0002]

2. Description of the Related Art With the progress of genetic engineering, it has become necessary to rapidly decode DNA or RNA containing genetic information. For example, four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T) are arranged on DNA, and genetic information is determined by the sequence of these bases. Therefore, vigorous research has been conducted on the relationship between the expression of a gene in a living body and the nucleotide sequence on DNA. For that purpose, it is necessary to rapidly determine the base sequence on DNA, and several determination methods have been proposed to date (“Cell Engineering” Vol. 1, No. 1, No. 2 (19).
82)).

The most widely used of these proposed methods is the method of determining the NDA nucleotide sequence by the Maxam-Gilbert method described below. In this method, it is determined by the following steps.

(1) First, the DNA fragment to be sequenced is separated.

(2) Both ends (5 'ends) of the separated DNA fragment are labeled with radioactive phosphorus ( ³² P).

(3) Separate the double strands of the DNA fragment labeled at both ends to prepare a single-stranded DNA fragment labeled with ³² P at only one end. Alternatively, a DNA fragment labeled at both ends is cleaved with a restriction enzyme, and then the DNA fragment is separated to prepare a DNA fragment labeled with ³² P at only one end.

(4) The above-obtained DNA fragment labeled at one end with ³² P is added to the base G on the DNA.
And then a chemical substance for cleavage is allowed to act to cleave at the modified base G site.
Cleavage should occur on average once for each DNA fragment. By doing this, as shown in FIG.
DNA fragments of various lengths containing one end labeled with ³² P and the other end cleaved at the site of base G are obtained. In this case, as shown in FIG. 1 (C), a DNA fragment labeled with ³² P and not containing one end can be obtained at the same time. However, since the radiation emitted from ³² P is measured as described later, these DNA fragments are damaged. do not become.

(5) The same operation is individually performed for the other bases A, C and T.

Regarding the base A and the base T, there is no suitable chemical substance that acts specifically on each of these sites.
Therefore, for the cleavage of the base A site, for example, a chemical substance that acts on both the base G and the base A is used to perform the cleavage treatment (G + A) at the base G site and the base A site to obtain the base G When there is no pattern of the DNA fragment cleaved at the site A, the DNA fragment cleaved at the site A is determined. Similarly, in the case of cleavage of the base T site, for example, a chemical substance that acts on both the base C and the base T is used to perform a cleavage treatment (C + T) at the base C site and the base T site, When the pattern of the DNA fragment cleaved at the C site does not exist, the DNA fragment cleaved at the base T site is used. Thus one end
4 types of D labeled with ³² P and cleaved at the bases G, G + A, C + T, C at the other end
Obtain a group of NA fragments.

(6) These DNA fragments are arranged for each kind on a single electrophoresis plate (not shown) and simultaneously run in a gel as a migration medium.

(7) After electrophoresis for an appropriate time, a photographic plate is brought into close contact with the gel and exposed to ³² P radiation. On the photographic plate, four strip-shaped patterns corresponding to the fragments cut by the bases G, G + A, C + T and C are obtained. The migration rate of DNA fragments differs depending on the number of bases. The shorter the number of bases and the shorter the number, the faster the migration.

(8) Finally, the bases G, G + A, C + T,
When the fragment cleaved by C is read from the shorter side, the nucleotide sequence on the DNA can be sequentially determined from the end side labeled with ³² P.

[0013]

This method requires the use of radioactive phosphorus, the electrophoresis can be performed in several hours, but the exposure to the photographic plate takes about one day and night, and about 200 to 300 at a time. There was a problem such as the inconvenience of being able to determine only up to the base level.

In view of the above problems, an object of the present invention is to provide an apparatus capable of easily determining a base sequence in a short time.

[0015]

[Means for Solving the Problems] Is the above-mentioned object a nucleic acid sample?
For each of the multiple sample fragments generated from
A nucleic acid fragment group obtained with, each base at a site different in cut to length, and cut the plurality of kinds of nucleic acid fragments set labeled with a different fluorescent substance for each type of base sites, the sample Different for each fragment
That a gel electrophoresis plate in which a plurality have a migration path for electrophoresis is injected into position, the electrophoretic force was applied in one direction to nucleic acid fragments group injected by applying an electric field to the gel electrophoresis plate, it has been gel electrophoresis nucleic acid fragments by the type of light detected by the defined position of the electric field applying means allowed to migrate a plurality of types of nucleic acid fragments group along a plurality of electrophoretic paths which are formed in the electrophoresis plate, said plurality of migration path By detecting the passage of a nucleic acid fragment through the limited position while identifying the type of
This is achieved by a configuration for determining the base sequence of a nucleic acid sample.

[0016]

According to the above construction, since the base sequence is determined by the time change of a plurality of detection outputs obtained while continuing the electrophoresis, it is not necessary to pay attention to the time for stopping the electrophoresis as in the conventional case. , Always high resolution is obtained. Furthermore, since it is not necessary to transfer the pattern to the photographic plate, it takes time to handle the gel and time for transfer, and it is repeatedly electrophoresed with different migration times depending on the range of the length of the fragment to be separated and measured. No need to
It can measure continuously from short fragments to long fragments. Furthermore, a large number of samples can be electrophoresed simultaneously using a single gel electrophoresis plate. That is, the time for determining the base sequence can be greatly reduced.

[0017]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIGS.

First of all, the DNA to be measured is divided into several fragments. This division of DNA is performed using a restriction enzyme capable of recognizing and cleaving a specific nucleotide sequence site on the DNA. Some of these restriction enzymes recognize and cleave a plurality of (4, 6 etc.) base sequences. The fragments thus formed are called F ₁ , F ₂ , ... F _n . The challenge is to determine the base sequences of these DNA fragments. For this sequencing, each DNA is further cleaved into shorter fragments. In order to avoid confusion, DNA fragments F ₁ , F ₂ , ...
Each _n is called a sample DNA.

Next, after separating and purifying each sample DNA,
Each is divided into four pieces, and a DNA fragment whose one end is labeled with a fluorescent substance is prepared from the collected sample DNA.

Next, four kinds of bases G, G + A, C + T, C
Is modified with a chemical substance that specifically or selectively modifies it, and then the modified site is partially cleaved with a chemical substance that selectively cleaves.

Of the fragments prepared from the sample DNA represented by F ₁ above, the _one in which the site of base G is partially cleaved is called F _1G . Among the fragments F _1G , there are a fragment labeled at one end with a fluorescent substance and the other end cleaved at various sites of the base G, and an unlabeled fragment having both ends cleaved at various sites of the base G. included. Focusing only on those labeled at one end, a set of large and small fragments with one end labeled and the other end cleaved once at various base G sites is formed. Similarly bases G + A, C + T, C
You can also set large and small fragments of.

Next, these four types of fragments F _1G ,
The set of F _{1G + A} , F _{1C + T} , and F _1C is injected into the electrophoresis tank for separation. The large and small fragments are separated because their migration speeds differ depending on their length, and those of the same length migrate at the same speed. In the conventional method, the base sequence was determined by transferring the band using a photographic plate after sufficient electrophoresis and analyzing the pattern. In the present invention, while the migration is continued, it is detected by the emission of the fluorescent substance that the fragment has passed through the specific location on the migration path.

FIG. 2 shows a base sequence determination device equipped with an electrophoresis tank, a detector according to the present invention, a data processing device, an output device and the like. Positive and negative electrodes 2A and 2B are arranged at both ends of the electrophoresis tank 1, and a migration driving power source 3 for applying a voltage between both electrodes 2A and 2B is installed. Electrophoresis tank 1
A gel electrophoresis plate 4 is provided on the upper surface of the. Detectors at four predetermined positions on the gel electrophoresis plate 4 on the migration paths of four kinds of fragments F _1G , F _{1G + A} , F _{1C + T} , and F _1C whose one end is cleaved with the bases G, G + A, C + T, and C. 5 (a), 5
Provide (b), 5 (c), and 5 (d). The number of detectors is 4
The number is not limited to five, and may be five. In this case, one is used for reference. Further, four may be provided as one set and a plurality of sets may be provided, or five may be provided as one set and a plurality of sets may be provided. This detector is a photodetector that detects fluorescence from the fluorescent substance at one end of the DNA fragment. Here, the kind of light is changed, that is, four kinds of fragment groups (F
_1G , F _{1G + A} , F _{1C + T,} and F _1C ), the type of the fluorescent substance to be labeled is changed, and the types of fragments (F _1G , F _{1G + A} , F _{1C + T,} and F _{1C + T} and F _1C ) so that one sample DNA (F ₁ , F ₂ , ...
Swim along the migration path with one detector for each _n )
Nucleotide sequences emitted from moving fluorescently labeled DNA fragments
The detection output necessary for the determination can be obtained.

In this way, one detector may detect that a plurality of types of fragments have passed. These detectors 5 (a), 5 (b), 5 (c) and 5 (d) detect each fragment passing through while migrating and output a detection signal. The state will be described with reference to FIG.

In FIG. 3, each fragment F _1G , F
Detectors 1 (a), 5 on the migration path of _{1G + A} , F _{1C + T} , F _1C
The detection signals from (b), 5 (c), and 5 (d) are the amplifier 6
Is amplified by. The amplified signal is amplified by the amplifier 6 over time.
Is transmitted as a signal as shown in FIG. 3 (b). The peak portion of the signal intensity is detected by the detectors 5 (a), 5
(B), 5 (c) and 5 (d) are bases G and G +, respectively.
It shows that migration of A, C + T, and C fragments was detected. Alternatively, when the fluorescent substance is changed according to the type of fragment, the migration of each fragment can be similarly detected by the peak of each output wavelength-discriminated.

Next, this amplified signal is input to the data processing device 7 shown in FIG. 2 and decoded to determine the base sequence. In the signal shown in FIG. 3 (b), the peak located in the lower part of the figure indicates a fragment that migrated quickly in a short time, which means that a DNA fragment having a short length and a small molecular weight was detected. . Therefore, since the detection time differs each time the length changes by one base, the base sequence can be determined by sequentially reading the peaks that appear in a short time. For example, in the signal shown in FIG. 3 (b), the signal from the line of the fragment F _1G comes out first, so the terminal is the base G, and then the bases A, G, C, and
The sequence is sequentially determined as A, T, and C. After these outputs are organized in the data processing device 7, the output device 8, for example a printer, outputs them in characters such as GAGCATC in the order of arrangement.

As a nucleic acid base sequence determination device,
It suffices to have means for recording the time change of the output of each detection means (or each output for which the wavelength has been identified), and the above-mentioned amplifier, data processing device, output device, etc. can be provided as desired.

[0028]

The present invention has the following effects.

(1) It is not necessary to take a photograph of an electrophoretic pattern as in the prior art, and the base sequence can be determined in a short time while continuing the electrophoretic migration.

(2) In the conventional method, great care must be taken in the time required for electrophoresis. That is, if the time is too short, the DNA fragments will not be sufficiently separated, and at most about 100 bases can be decoded. If the time is too long, the small DNA fragments will reach the end of the gel and become unreadable. Therefore, it took time and effort to divide the gel into several layers for electrophoresis. According to the present invention, it is possible to measure from a small fragment to a fragment as large as the limit of the resolution by gel. Moreover, many samples can be electrophoresed on one gel.

(3) The above can also be said for RNA.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a method for preparing fragments for electrophoresis.

FIG. 2 is a diagram showing an embodiment of the present invention.

FIG. 3 is a diagram showing a time change of signals output from a detector and an amplifier in FIG.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Electrophoresis tank, 2A, 2B ... Electrode, 3 ... Electrophoresis drive power source, 4 ... Gel electrophoresis plate, 5 (a), 5 (b), 5
(C), 5 (d) ... Photodetector, 6 ... Amplifier, 7 ... Data processing device, 8 ... Output device.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // C12M 1/00 Z C12N 15/09 G01N 27/26 315 A 9050-4B C12N 15/00 A (72) Inventor Fumio Hishinuma, No. 11 Minami-Otani, 11 Machida, Tokyo 2 916, Mitsubishi Kasei Life Sciences Co., Ltd. (72) Inventor Tadayoshi Shiba, No. 11, Minami-Otani, 11 Machida, Tokyo 2 Co., Ltd. (56) Reference JP-A-60-220860 (JP, A) JP-A-63-231247 (JP, A) JP-A 64-1960 (JP, A)

Claims (1)

[Claims] 1. A plurality of sample cells produced from a nucleic acid sample.
Multiple types of nucleic acids obtained for each of the fragments
Is a fragment group, different types of terminal positions base, the plural kinds of nucleic acid fragments set labeled with a different fluorescent substance for each type of base of the terminal position is the upper
Each sample fragment is injected at a different position and migrates
A gel electrophoresis plate a plurality have a migration path, applying an electric field to the gel electrophoresis plate electrophoretic force imparted in one direction to the injected the nucleic acid fragment group, the formed in the gel electrophoresis plate with While distinguishing the type of nucleic acid fragment by the electric field applying means for migrating the plurality of kinds of nucleic acid fragment groups along the plurality of migration paths and the kind of light detected at the limited positions of the plurality of migration paths. Nucleic acid fragment has passed through the limited position
And a light detecting means for detecting, bases of nucleic acids and determining the nucleotide sequence of the nucleic acid trial <br/> charge by the time change of the detection output of each type of light obtained from the light detecting means Sequencer.