JPH0610665B2 - Nucleic acid nucleotide sequencer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Use of the Invention] The present invention relates to DNA (deoxyribonucleic acid) or RNA.
The present invention relates to a device for determining a base sequence in a nucleic acid such as (ribonucleic acid).

[Background of the Invention]

With the progress of genetic engineering, it has become necessary to rapidly decode DNA or RNA containing genetic information. For example, on DNA, adenine (A), guanine (G),
Four types of bases, cytosine (C) and thymine (T), are arrayed, and genetic information is determined by the sequence of these bases. Then, the relationship between the expression of a trait in a living body and the base sequence on DNA is vigorously studied. For that purpose, it is necessary to quickly determine the base sequence on DNA, and several determination methods have been proposed to date (“Cell Engineering” Vol.1, NO.1, NO.2 (1982)). .

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

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

(2) Label both ends (5 'ends) of the separated DNA fragment 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) DN obtained at the above with one end labeled with ³² P
A chemical substance that modifies the base G on DNA is allowed to act on the A fragment, and then a chemical substance for cleavage is allowed to act,
Cleave at the site of modified base G. Cleavage should occur on average once for a DNA fragment. As a result, as shown in FIG. 1 (b), DNA fragments of various lengths containing one end labeled with ³² P and the other end being cleaved at the site of base G are obtained. In this case, Fig. 1
As shown in (c), a DNA fragment labeled with ³² P and not containing one end can be obtained at the same time, but these do not cause an obstacle because the radiation emitted from ³² P is measured as described later.

(5) Do the same for each of the other bases A, C, T individually.

As for 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, If there is no pattern of DNA fragment cleaved at C site, DN cleaved at base T site
A fragment. Thus one end is labeled with ³² P and the other end is base G, G + A, C +, respectively.
A group of 4 DNA fragments cleaved at the T and C sites is obtained.

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

(7) After electrophoresis for an appropriate period of 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. DN
The migration rate of the A fragment differs depending on the number of bases. The shorter the number of bases, the faster the migration, so
Fast transition from one end to the other.

(8) Finally, by reading the fragments cut by the bases G, G + A, C + T, and C from the shorter side, the base sequence on the DNA can be sequentially determined from the end side labeled with ³² P.

This method requires the use of radioactive phosphorus, the electrophoresis can be performed in a few hours, but it takes about one day to expose the photographic plate to light, and the inconvenience that only about 200 to 300 bases can be determined at a time. There were problems such as being there.

[Object of the Invention]

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.

Another object of the present invention is to provide an apparatus capable of further improving the base sequence determination accuracy.

[Outline of Invention]

A feature of the present invention is that a gel electrophoresis plate is labeled with a fluorescent substance, and a plurality of types of nucleic acid fragment groups having different types of bases at the cleavage site are individually separately injected, and an electric field is applied to the gel electrophoresis plate. An electric field applying means for applying an electrophoretic force to the injected nucleic acid fragment group in one direction so as to individually migrate the plural kinds of nucleic acid fragment groups along a plurality of migration paths formed in the gel electrophoresis plate. And a photodetection unit for individually detecting passage of the nucleic acid fragment at each limited position of the plurality of migration paths, and the time of detection output for each of the plurality of migration paths obtained from the light detection unit. The configuration is such that the base sequence of the target sample is determined by the change.

According to the above configuration, 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, and it is always high. Resolution is obtained. Further, since it is not necessary to transfer the pattern to the photographic plate, the labor for handling the gel and the time for transfer can be saved. In particular, it is possible to perform continuous separation and measurement from short fragments to long fragments without using the technique of repeating electrophoresis and transcription at different migration times according to the range of the length of the fragments to be separated and measured. The time can be greatly reduced.

Example of Invention

An embodiment of the present invention will be described below with reference to FIGS. 2 and 3.

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 fragment thus formed is F ₁ ,
Called F ₂ , ... F _n . The challenge is to determine the base sequences of these DNA fragments. As described later, F _1, F 2 is the target for sequencing in order to avoid confusion since obtaining a shorter electrophoretic fragment group for this sequencing is referred to as a _... F _n with sample DNA.

Next, after separating and purifying each sample DNA, the DN having one end labeled is treated according to the above-mentioned conventional method.
Prepare the A fragment. However, as a substance that labels, radioactive phosphorus ³² rather than P, a fluorescent substance is used.

Next, for each sample DNA, the labeled sample is divided into four, and different adjustments are performed for each division. That is, each of the four divided samples is individually modified with a chemical substance that specifically or selectively modifies the four bases G, G + A, C + T, and C, and then the modified site is selectively cleaved. Partially cut by chemical substances.

Of the fragments prepared from the sample DNA represented by F ₁ above, the site of base G is partially cleaved and is referred to as 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, for the bases G + A, C + T, and C, large and small fragment sets can be set.

Next, these four types of fragments F _1G , F _{1G + A} , F
Separation is performed by individually injecting the set of _{1C + T} and F _1C at different positions along one end of the electrophoresis plate. 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,
Here, 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, the passage of the fragment through the specific location on the migration path is detected by the fluorescence from the fluorescent substance.

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 2 are provided at both ends of the electrophoresis tank 1.
A and 2B are arranged, and an electrophoretic drive power source 3 for applying a voltage between both electrodes 2A and 2B is installed. On the upper surface of the electrophoresis tank 1,
A gel electrophoresis plate 4 is provided. One end is base G, G +
Four kinds of fragments F _1G , F _{1G + A} , F _{1C + T} , and F _1C cleaved by A, CC + T, and C are near one edge of the gel electrophoresis plate 4, more specifically, near the negative electrode 2B. Individual injection is performed at four injection positions (not shown) arranged side by side. Then, due to the electric field generated between the electrodes 2A and 2B, each of the segment groups migrates in the gel along the four migration paths from the bottom to the top in FIG. Gel electrophoresis plate 4 on these migration paths and at the same distance from the injection position
Detectors 5 (a), 5 (b), 5 at four predetermined positions of
(C) and 5 (d) are provided. The number of detectors is not limited to four, but may be five, in which 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. In addition, the kind of light is changed, that is, four kinds of fragment groups (F _1G ,
If the types of fluorescent substances to be labeled are changed among F _{1G + A} , F _{1C + T,} and F _1C ) so that the types of fragments can be discriminated by the fluorescence wavelength, one sample DNA (F
_One detector for each of F ₁ , F ₂ , ..., F _n ). In this way, one detector may detect the passage of multiple types of fragments. These detectors 5
(A), 5 (b), 5 (c), and 5 (d) detect each fragment passing while migrating and output a detection signal. The state will be described with reference to FIG.

In FIG. 3, each fragment F _1G , F _{1G + A} ,
Detectors 5 (a), 5 (b) on the migration path of F _{1C + T} , F _1C ,
The detection signals of 5 (c) and 5 (d) are amplified by the amplifier 6. The amplified signal is transmitted from the amplifier 6 to the third signal over time.
It is sent out as a signal as shown in (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, G + A, C + T,
It shows that the migration of the C fragment was detected.

Next, this amplified signal is input to the data processing device 7 shown in FIG. 2 and decoded to determine the base sequence. Third
In the signal shown in (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, regarding 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, A, T, C. The sequence is determined sequentially. These outputs are arranged in the data processing device 7, and then output by the processing device 8, for example, a printer, in characters such as GAGCATC, in the order of arrangement.

It should be noted that the nucleic acid base sequence determination device may be provided with a unit for recording the time change of the output of each photodetector, and the above-mentioned amplifier, data processing device, output device, etc. may be provided as desired.

〔The invention's effect〕

 The present invention has the following effects.

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

(2) In the conventional method, great care was required in the time required for electrophoresis. That is, if the time is too short
Since the fragments are not separated, only about 100 bases can be decoded at most, and if the time is too long, a small DNA fragment reaches the end of the gel and becomes 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.

(3) The above also applies to RNA.

(4) In the configuration in which a migration route is provided for each fragment group corresponding to each type of base and a photodetection unit is arranged, the difference between the migration conditions and the time detection conditions for each group can be reduced, so that the mutual arrangement of each base is It can be accurately understood and the sequencing accuracy can be improved.

[Brief description of drawings]

FIG. 1 (a) is a diagram schematically showing a DNA fragment labeled at one end. FIG. 1 (b) is a diagram schematically showing a DNA fragment in which the fragment shown in FIG. 1 (a) is further cleaved at the base G site by a reaction specific to the base G. FIG. 1 (c) shows D shown in FIG. 1 (b).
It is the figure which showed the fragment except NA fragment. FIG. 2 is a diagram showing an embodiment of the present invention. Third
The figure is a diagram showing the signals output from the detector and the amplifier in FIG. 1 ... Electrophoresis tank, 2A, 2B ... Electrode, 3 ... Migration driving power source, 4 ... Gel electrophoresis plate, 5 (a), 5 (b),
5 (c), 5 (d) ... Detector, 6 ... Amplifier, 7 ...
Data output device, 8 ... Output device.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical indication location G01N 33/58 A 7055-2J (72) Inventor Fumio Hishinuma 11-11 Minamiootani, Machida, Tokyo Address 2 Mitsubishi Kasei Life Science Research Institute Co., Ltd. (72) Inventor Tadayoshi Shiba No. 11 Minami Otani, Machida City, Tokyo 916 Address 2 Mitsubishi Kasei Life Science Research Institute Co., Ltd.

Claims (1)

[Claims] 1. A gel electrophoresis plate, which is labeled with a fluorescent substance and in which a plurality of types of nucleic acid fragment groups having different types of bases at the cleavage site are individually and separately injected, and an electric field is applied to the gel electrophoresis plate for injection. An electric field applying means for applying an electrophoretic force in one direction to the nucleic acid fragment group thus formed, and individually migrating the plurality of nucleic acid fragment groups along a plurality of migration paths formed in the gel electrophoresis plate; A time change of the detection output for each of the plurality of migration paths obtained from the light detection means, the light detection means individually detecting passage of the nucleic acid fragment at each limited position of the plurality of migration paths. An apparatus for determining a base sequence of a nucleic acid, characterized in that the base sequence of a target sample is determined by.