JPS60161559A - Apparatus for determining arrangement of base of nucleic acid - Google Patents

Abstract

PURPOSE:To enable the determination of an arrangement of bases in a short time with no need to photograph a migration pattern while the migration continues by a method wherein fragments of a nucleic acid cut in various lengths in the part of each base are made to migrate at a speed in accordance with a molecular weight, and detectors are provided on the route of migration. CONSTITUTION:A nucleic acid is cut into fragments in various lengths in the part of each base, and the end of each fragment is labeled with <32>P or a fluorescent material. A separation plate 4 is provided on the top of an electrophoretic bath 1 with positive and negative electrodes 2A and 2B disposed in the opposite ends, and a radiation or a fluorescence is detected from four kinds of fragments thus labeled cut in bases G, G+A, C+T and C at the ends respectively, for instance, on a migration route by detectors 5(a), 5(b), 5(c) and 5(d) provided at four prescribed positions on the separation plate. Detection signals obtained thereby are sent to an amplifier 6, amplified signals are inputted to a data processing device 7, and data on an interpreted arrangement of the bases are outputted, in a character of GACAT or the like, to an output device 8. In this way, small and large fragments in a wide range can be measured in sufficient separation from one another in a short time.

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention is directed to the use of DNA (deoxyribonucleic acid) or RNA.
The present invention relates to a device for determining the base sequence of a nucleic acid such as (liponucleic acid).

[Background of the invention]

With advances in genetic engineering, it has become necessary to quickly decode DNA or RNA containing genetic information. For example, four types of bases, adenine (A), quanine (G), cytosine (0), and thymine (T), are arranged on DNA, and genetic information is determined by the sequences of these bases. Therefore, intensive research is being conducted on the relationship between trait expression in living organisms and base sequences on DNA. For that purpose D
It is necessary to quickly determine the nucleotide sequence on NA, and several determination methods have been proposed to date (Cell Engineering J Vow, 1, N11, N12 (198
2) ).

Among these proposed methods, the most widely used method is the Maxapn-GHbert method described below for determining DNA base sequences. In this method, the following steps determine:

(1) List of 7 lagmen of DNA to be sequenced , and are first separated.

(2) Label both ends of the separated DNA fragment with radioactive phosphorus (32P).

(3) Separate the double strands of the DNA 7 fragment labeled at both ends to prepare a single-stranded DNA fragment with only one end labeled with 32p. Restrict the DNA fragment labeled at 0 or both ends. After cutting with an enzyme, the DNA fragments are separated and only one end is 32p.
Prepare a labeled DNA fragment.

(4) The DNA fragment obtained above, labeled with 32p at one end, is treated with a chemical substance that modifies the base G on the DNA, and then with a chemical substance for cleavage to produce the modified base. Cut at site G. Cut each D
Let average-recurrence occur for the NA fragment. In this way, as shown in FIG. 1(b), DNA7 fragments of various lengths containing one end labeled with 32p and the other end cleaved at the base G site are obtained. In this case, as shown in Fig. 1 (C), DNA fragments labeled with 32p that do not contain the two ends are also obtained at the same time, but as will be described later, since the radiation emitted from 32p is measured, these fragments may be a problem. No.

(5) Do the same thing individually for other bases A%O and T.

Note that for base A and base T, there are no suitable chemical substances that act specifically on these sites. Therefore, to cleave the base A site, for example, using a chemical substance that acts on both base G and base A, cleavage treatment (G0A) is performed at the base G site and the base A site, A case where there is no pattern of DNA fragments cut at the base G site is defined as a DNA fragment cut at the base A site. Similarly, in the case of cleavage at the base T site, for example, a chemical substance that acts on both base C and base T is used to perform cleavage treatment (0+T) at the base O site and the base T site, and the base If there is no pattern of DNA fragments cut at site C, DNA cut at site T.
Let it be A fragment. Thus, one end is labeled with 32p, and the other end is labeled with the bases G, G0A, O+, respectively.
Four groups of DNA7 fragments cleaved at the T and 0 sites are obtained.

(6) These DNA fragments are arranged for each type on a single electrophoresis plate (not shown) and electrophoresed simultaneously.

(7) After electrophoresis for an appropriate time, a photographic plate is brought into close contact with the gel and exposed to 32p radiation.

On the photographic plate, a four-band pattern corresponding to the fragments cleaved with the bases G, G0A, 0+T, and O is obtained. DNA fragments have different migration speeds depending on their number of bases. The fewer and shorter bases they have, the faster they migrate, and therefore the faster they migrate from one end to the other.

(8) Finally, reading the fragments cleaved at the bases G, G0A, o+'r, and O from the shortest one,
The base sequence on the DNA can be sequentially determined starting from the end labeled with 32p.

This method requires the use of radioactive phosphorus, electrophoresis can be carried out in a few hours, but exposing it to a photographic plate takes about day and night, and it is inconvenient that only about 200 to 300 bases can be determined at a time. There were problems such as the presence of

[Purpose of the invention]

SUMMARY OF THE INVENTION In view of the above-mentioned problems, an object of the present invention is to provide an apparatus that can easily determine base sequences in a short time.

[Summary of the invention]

In order to achieve the above object, the present invention is characterized by a tank into which 72 fragments of nucleic acid cleaved to various lengths at each base site are injected, and A nucleic acid base sequencing apparatus comprising means for moving within the tank at a speed corresponding to the molecular weight of the nucleic acid, and means for detecting the 7 fragments of the nucleic acid provided on the movement path of each of the 72 fragments of the nucleic acid. .

Other features of the present invention include a tank into which 72 fragments of nucleic acid cut into various lengths at each base site are injected, and means for moving the fragments within the tank at a speed corresponding to their molecular weight. , a nucleic acid base sequencing apparatus comprising means for detecting the fragments provided on each movement path of the seven fragments, and means for decoding the base sequence of the fragments based on a detection signal from the detection means. be.

[Embodiments of the invention]

An embodiment of the F1 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 called
This is done using a restriction enzyme that can recognize and cleave the specific base sequence site above.

Some of these restriction enzymes recognize and cleave multiple (4, 6, etc.) base sequences. The fragments formed by crushing are labeled F, , F2. ...It is called Fn. The challenge is to determine the base sequences of these DNA fragments.

Next, after separating and purifying each DNA fragment, it is treated according to the conventional method described above to prepare a DNA fragment having one end with 32p or 2 bells with a fluorescent substance.

Next, after modifying with a chemical substance that specifically or selectively modifies the four bases G, G+A, O+T, and Ot-,
Partial cleavage is performed using a chemical that selectively cleaves the modified site.

Of the above seven fragments F1, the one partially cleaved at the base G site will be referred to as FIG. Fragmen) F
IG includes seven fragments in which one end is labeled and the other end is cleaved at various sites on base G, and an unlabeled fragment in which both ends are cleaved at various sites on base G. Among these, if we focus on only those labeled at one end, a set of seven large and small fragments is formed, with one end labeled and the other end cleaved once at various base G sites. Similarly, the bases G+A, 0+T.

For C, a set of 7 large and small fragments can be created.

Next, these 4' types of 7 fragments F□. .

The set of FIG+A l ”IC+T I FIC is injected into an electrophoresis tank and separated. Large and small 77 segments have different migration speeds depending on their length, so they are separated, and those of the same length migrate at the same speed. .In the conventional method.

Here, after sufficient electrophoresis, the bands were transferred using a photographic plate, and the base sequence was determined by analyzing the pattern. In the present invention, while continuing electrophoresis, a radiation band or a band labeled with a fluorescent substance passing through a specific location on the electrophoresis path is detected.

FIG. 2 shows a base sequencing apparatus equipped with an electrophoresis tank, a detector, a data processing device, an output device, etc. according to the present invention. Positive and negative electrodes 2 are installed at both ends of the electrophoresis tank 1, respectively.
Electrophoresis drive power source 3 is installed to apply a voltage between both electrodes 2A and 2B. On the top surface of electrophoresis tank 1,
A separating plate 4 is provided. One end is base G, G0A, O+T
, four types of 7-ragment FIG + FI cleaved at O
G+A rFIC+TI Detectors 5 (a), 5 (b), 5 (
c) and 5(d) shall be provided. The number of detectors is not limited to four,
In some cases, there may be five pieces), in which case one piece is used for reference. Further, a plurality of sets of four may be provided, or a plurality of sets of five may be provided.

This detector is a radiation detector if the label at one end of the DNA7 fragment is 32p, or a photodetector if the label is a fluorescent substance. It should be noted that one detector may be sufficient as long as the intensity of the radiation or the type of light is changed so that the various types of seven fragments can be identified. This single detector may detect the passage of multiple types of 7 fragments. These detectors 5
(a), 5(b), 5(c), and 5(d) detect each of the seven fragments that pass while migrating, and output a detection signal.

The state will be explained based on FIG.

In FIG. 3, each fragment FIG I FIQ+
A IFIC + TI FIC detector 5 on the electrophoresis path (
b), 5 (0).

The detection signals from 5(c) and 5(d) are amplified by an amplifier 6. The amplified signal is transferred from the amplifier 6 to the third
(b) It is sent as a signal as shown in the figure. The peak portion of the signal intensity is at each detector 5 (a).

5(b), 5(c), and 5(d) indicate that migration of fragments of bases G, G0A, O+T, and O was detected, respectively.

Next, this amplified signal is input to the data processing device 7 shown in FIG. 2, where it is decoded and the base sequence is determined. Third (
b) Among the signals shown in the figure, the peak located at the bottom of the figure indicates a fragment that migrated quickly in a short time, meaning that a DNA fragment with a short length and 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 fragment F1o comes out first, so the terminal is base G, then base A.

The sequence is determined in sequence: G, O, A, T, O.

After these outputs are organized in the data processing device 7, they are sent to the GAGO by an output device 8, for example, a printer, in the order of arrangement.
It is output in characters such as ATO.

Note that the nucleic acid base sequencing apparatus only needs to be equipped with a detection means, and the above-mentioned amplifier, data processing device, output device, etc. can be provided as desired.

〔Effect of the invention〕

According to the present invention, there are the following effects.

(1) There is no need to take photographs of electrophoresis patterns as in the past, and base sequences can be determined in a short time while continuing electrophoresis.

(2) In the conventional method, great care was required regarding the time required for electrophoresis. In other words, if the time is too short, the DN is insufficient.
Since the A fragment is not separated, only about 100 bases can be decoded at most, and if the time is too long, the small DNA7 fragment will reach the end of the gel and become unreadable. Therefore, it was time consuming to perform the electrophoresis in separate stages. According to the present invention, it is possible to measure fragments ranging from small fragments to fragments so large that they reach the limit of the separation ability of gels.

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

[Brief explanation of drawings]

FIG. 1(a) is a diagram schematically showing a DNA fragment labeled at one end. Figure 1(b) shows the base G
1(a) 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 . FIG. 1(C) is a diagram showing fragments other than the DNA fragment shown in FIG. 1(b). Figure 2 shows
FIG. 1 is a diagram showing an embodiment of the present invention. FIG. 3 is a diagram showing signals output from the detector and amplifier in FIG. 2. 1... Electrophoresis tank, 2A, 2B... Electrode, 3...
Electrophoresis drive power source, 4... separation plate, 5 (a), 5 (b),
5 (c). 5 (Nil...detector, 6...amplifier, 7...data processing device, 8...output device. Spear 1 Figure■---- 2nd mouth 3rd eye

Claims (1)

[Scope of Claims] 1. A cypress into which nucleic acid fragments cut into various lengths at each base site are injected, and means for moving the fragments within the tank at a speed corresponding to their molecular weight; A nucleic acid base sequencing apparatus, comprising: means for detecting the fragment, which is provided on a movement path of the fragment. 2. Nucleic acid base sequencing according to claim 1, wherein the tank is an electrophoresis tank, and the moving means is a migration drive power source that applies electrophoresis force to the fragments. Device. 3. The nucleic acid base sequencing apparatus according to claim 1, wherein the 7 fragments are doped with a radioactive substance, and the detection means is a radiation detector. 4. The fragment is labeled with a fluorescent substance,
2. The nucleic acid base sequencing apparatus according to claim 1, wherein the detection means is a photodetector. 5. The nucleic acid base sequencing apparatus according to claim 1, wherein four or more detection means are provided. 6. The nucleic acid base sequencing apparatus according to claim 1, wherein one of the detection means detects a plurality of types of the fragments. 7. A tank into which 7 fragments of nucleic acid cut into various lengths at each base site are injected, means for moving the 7 fragments within the tank at a speed corresponding to their molecular weight, and movement of the fragments. 1. A nucleic acid base sequence determining apparatus, comprising: means for detecting the fragments provided on a path; and means for decoding the base sequence of the fragments based on a detection signal from the detecting means. 8. Nucleic acid base sequencing according to claim 7, wherein the tank is an electrophoresis tank, and the moving means is a migration drive power source that applies electrophoresis force to the fragments. Device. 9. 8. The nucleic acid base sequencing apparatus according to claim 7, wherein the fragment is doped with a radioactive substance, and the detection means is a radiation detector. 10. The nucleic acid base sequencing apparatus according to claim 7, wherein the fragment is labeled with a fluorescent substance, and the detection means is a photodetector. 11. The nucleic acid base sequencing apparatus according to claim 7, wherein four or more detection means are provided. 12. The nucleic acid base sequencing apparatus according to claim 7, wherein the one detection means detects a plurality of types of the fragments.