JPH08211017A - Dna base sequence determining apparatus - Google Patents
Dna base sequence determining apparatusInfo
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- JPH08211017A JPH08211017A JP7305505A JP30550595A JPH08211017A JP H08211017 A JPH08211017 A JP H08211017A JP 7305505 A JP7305505 A JP 7305505A JP 30550595 A JP30550595 A JP 30550595A JP H08211017 A JPH08211017 A JP H08211017A
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- Prior art keywords
- electrophoretic
- light
- dna
- scanning
- fluorescence
- Prior art date
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は電気泳動装置に係り、特
にＤＮＡ上の塩基配列決定装置（ＤＮＡシーケンサー）
として使用するに好適な電気泳動装置に関するものであ
る。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoretic device, and more particularly to a DNA base sequencer (DNA sequencer).
The present invention relates to an electrophoretic device suitable for use as.
【０００２】[0002]
【従来の技術】従来、ＤＮＡ上の塩基配列の決定にはラ
ジオアイソトープを用いた方法が用いられていたが、塩
基を蛍光ラベルして光学的に検出する手法の開発が望ま
れている。これを実現するために図４に示すように、特
定末端を持ち片方の末端が、アデニン塩基（Ａ）、グア
ニン塩基（Ｇ）、シトシン塩基（Ｃ）、あるいはチミン
塩基（Ｔ）であるＤＮＡ断片を別々の泳動路上を泳動さ
せ、光を照射して泳動路毎に設けられた光電子増倍管に
より泳動ＤＮＡ断片を計測する手法が提案されている。
（昭和５８年度科研費補助金（総合研究（Ａ））研究成
果報告集Ｐ．２０〜２５）また、末端塩基種の異なる４
種の断片群をそれぞれ蛍光波長の異なる蛍光体でラベル
し、カラム状のゲル中を泳動させ、光を照射して得られ
る発光を回折格子で分光し４個の光電子増倍管で検出す
る方式も提案されている。2. Description of the Related Art Conventionally, a method using a radioisotope has been used to determine a base sequence on DNA, but it is desired to develop a method for optically detecting a base by fluorescent labeling. To achieve this, as shown in FIG. 4, a DNA fragment having a specific end and one end being an adenine base (A), a guanine base (G), a cytosine base (C), or a thymine base (T) Have been proposed in which electrophoresis is performed on separate electrophoresis paths, and light is irradiated to measure electrophoretic DNA fragments using photomultiplier tubes provided for each electrophoresis path.
(Fiscal 1983 Grant-in-Aid for Scientific Research (Comprehensive Research (A)) Research Results Report P.20-25)
A method in which a group of seed fragments is labeled with phosphors having different fluorescence wavelengths, migrated in a column-shaped gel, and the light emitted by light irradiation is dispersed by a diffraction grating and detected by four photomultiplier tubes. Is also proposed.
【０００３】[0003]
【発明が解決しようとする問題点】これらの提案されて
いる方法では、各ＤＮＡ断片群毎に光検出器を設ける必
要があり、一種の試料につき４ケの検出器が入用であ
る。光検出器には光電増倍管が用いられるが、平板型電
気泳動板を用いる場合、各泳動路上に光電増倍管が一直
線上に並ぶことになる。このため泳動路間隔は光電増倍
管のサイズで決定され、１０ｍｍが最小である。したが
って１つのＤＮＡ試料の解析には４泳動帯が必要で泳動
板の４ｃｍの巾を必要とする。[Problems to be Solved by the Invention] In these proposed methods, it is necessary to provide a photodetector for each DNA fragment group, and four detectors are required for one type of sample. Although a photomultiplier tube is used as the photodetector, when a flat plate type electrophoresis plate is used, the photomultiplier tubes are arranged in a straight line on each migration path. Therefore, the migration path interval is determined by the size of the photomultiplier tube, and 10 mm is the minimum. Therefore, analysis of one DNA sample requires four migration zones and a width of 4 cm of the migration plate.
【０００４】一方、ＤＮＡ塩基配列の迅速決定法として
普及してきているショットガン方式などでは目的とする
ＤＮＡを細断し、多種の小さな断片の配列決定を同時に
行なうものである。これを実行するには一度に多くの泳
動帯を活用できる事が必要である。しかし、上述のよう
に従来法では一板の泳動板（有効巾〜２０ｃｍ）上に高
々２０泳動帯、試料数にして５ヶの測定ができるだけで
あった。すなわち多種試料を同時解析する能力に欠ける
難点があった。[0004] On the other hand, in the shotgun method and the like, which have become widespread as a rapid method for determining a DNA base sequence, a target DNA is shredded and sequence determination of various small fragments is performed at the same time. To do this, it is necessary to be able to utilize many migration zones at once. However, as described above, according to the conventional method, it was possible to measure at most 20 electrophoretic bands on one electrophoretic plate (effective width of up to 20 cm) and to measure 5 samples. That is, there is a drawback that the ability to simultaneously analyze various samples is lacking.
【０００５】[0005]
【問題点を解決するための手段】本発明は上記問題点を
解決するためになされたものである。本発明では従来の
１泳動帯毎に検出器を設ける代わりに複数個の泳動帯に
一個の検出器を設置し、各泳動帯からの光を時分割して
検出している。得られた信号はある時間毎に異なる泳動
帯からのものとなるが、信号を選別サンプリングする事
により各泳動帯からの信号を区別してその時間変化を記
録できる。各泳動帯からの光を時分割し検出する手段と
しては、各泳動路に時分割して光を照射すること、ある
いは各泳動路には常に光を照射しておくが泳動路と検出
器の間に光の遮蔽部材を設けて各泳動路からの光を選択
的に検出器に伝達するように構成する。The present invention has been made to solve the above problems. In the present invention, one detector is installed in a plurality of electrophoretic zones instead of providing a detector for each electrophoretic zone in the related art, and the light from each electrophoretic zone is detected by time division. The obtained signals are obtained from different migration bands at certain times, but by selectively sampling the signals, the signals from each migration band can be distinguished and their time changes can be recorded. As means for time-division and detection of light from each migration zone, each migration path is time-divided with light, or each migration path is always irradiated with light. A light-shielding member is provided between them so that light from each migration path is selectively transmitted to the detector.
【０００６】[0006]
【作用】複数個の泳動帯に一個の検出器を設置し、各泳
動帯からの光を時分割して検出するように構成してお
り、検出器の数よりも泳動帯を多くすることができるた
め、検出器の設置場所の関係で制限されていた泳動帯の
設置数を増大させることができる。[Function] One detector is installed in a plurality of electrophoretic zones, and the light from each electrophoretic zone is time-divisionally detected to detect the number of electrophoretic zones more than the number of detectors. Therefore, it is possible to increase the number of electrophoretic bands installed, which was limited by the installation location of the detector.
【０００７】[0007]
【実施例】以下、本発明の一実施例を図１により説明す
る。レーザー光１は回転ミラー４などにより反射された
後ミラー２〜３により反射され電気泳動板を照射する。
回転ミラーは９０°ずつ回転し、入射光と４５°をなす
角で約１秒間静止する。静止面の位置により４つの光線
系路が選択できるようになっており、これらは照射光路
１４〜１７に相当する。照射光路１４は末端がＡで停止
している断片群の泳動路を照射し、１５〜１７は末端が
Ｔ、Ｇ、Ｃで停止する断片群の泳動路をそれぞれ照射す
る。このように回転ミラー４を動作させることにより照
射光路を変化させるが、ハーフミラーなどにより分割
し、４つづつのセットからなる複数の泳動路を照射する
ことができる。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIG. The laser beam 1 is reflected by the rotating mirror 4 and the like, and then reflected by the mirrors 2-3, and irradiates the electrophoretic plate.
The rotating mirror rotates by 90 ° and stands still for about 1 second at an angle of 45 ° with the incident light. Four light path paths can be selected depending on the position of the stationary surface, and these correspond to the irradiation light paths 14 to 17. The irradiation optical path 14 irradiates the migration paths of the fragment groups whose ends are stopped at A, and the irradiation paths 15 to 17 respectively irradiate the migration paths of the fragment groups whose ends are stopped at T, G, and C. Although the irradiation optical path is changed by operating the rotating mirror 4 in this way, it is possible to irradiate a plurality of migration paths made up of four sets by dividing by a half mirror or the like.
【０００８】光照射によって発せらせた蛍光はフィルタ
ー７を通過し検出帯８に入る。一方、励起光はフィルタ
ー７で除去される。検出器８には４つの泳動路からの蛍
光信号が交互に入ってくるが、同期回路１１を用いて泳
動路毎の信号を混合しないように積算してメモリー１２
に蓄える。メモリーとして二次元のものを考えると、横
軸に泳動帯の種類をとり、縦軸に時間あるいはサンプリ
ングＮｏ．を取る事ができる。各泳動帯からの蛍光強度
の時間変化をメモリーの縦列を読み出す事により知る事
ができる。４つの泳動帯で測定時間にずれがあり、これ
が問題となる場合には回転ミラーの回転周期を短かくす
れば良いが１〜４秒の周期で十分な事が多い。The fluorescence emitted by the light irradiation passes through the filter 7 and enters the detection zone 8. On the other hand, the excitation light is removed by the filter 7. The fluorescence signals from the four migration paths are alternately input to the detector 8, and the signals for each migration path are integrated by using the synchronization circuit 11 so that the signals are integrated so as not to mix.
To store. Considering a two-dimensional memory, the horizontal axis indicates the type of migration zone, and the vertical axis indicates time or sampling number. You can take The time change of the fluorescence intensity from each migration zone can be known by reading the column of memory. If there is a difference in measurement time between the four migration zones, and this is a problem, the rotation cycle of the rotating mirror may be shortened, but a cycle of 1 to 4 seconds is often sufficient.
【０００９】このようにして得られる信号の一例を図２
に示した。横軸は時間で（ａ）は泳動帯毎に現われる信
号１５’である。（ｂ）は検出器に流れる信号１６’を
示したもので時系列的にＡＧＣＴの信号が検出される。
（ｃ）は励起光が各泳動レーンを照射するタイミングを
示したものである。FIG. 2 shows an example of the signal thus obtained.
It was shown to. The horizontal axis represents time, and (a) is a signal 15 'that appears for each migration zone. (B) shows a signal 16 'flowing to the detector, in which AGCT signals are detected in time series.
(C) shows the timing at which the excitation light irradiates each migration lane.
【００１０】本実施例では回転ミラーを用いて光線を偏
向させたが、ビームをスキャンしたり、ピストン運動す
るミラーを用いても同様の作を行なうことができる。In this embodiment, the rotating mirror is used to deflect the light beam. However, the same operation can be performed by scanning the beam or using a mirror which moves the piston.
【００１１】上記実施例ではレーザー光を分割して利用
するために１つのレーン当りの照射光量が小さくなる難
点がある。図３はこの難点を解決するためになされた実
施例である。レーザー光１は側面からゲルに入射し、全
泳動帯の一定の箇所を照射する。蛍光は各泳動帯から発
せられるが、特定レーンの蛍光だけがスリット１８を通
過して検出器に到達できる。スリットはドライバー１９
により左右に移動でき、一定時間毎に異なる泳動帯から
の蛍光信号を通過させる事ができる。このスリットの移
動運動に同期して信号の積算が行なわれデータ処理され
る。この例では泳動パネルの両側に検出器を設け、スリ
ットの開閉を２段階ずつにし、泳動帯あたりの測定時間
をふやす事もできる。In the above embodiment, since the laser light is divided and used, the amount of irradiation light per lane is small. FIG. 3 shows an embodiment made to solve this difficulty. The laser light 1 is incident on the gel from the side surface and irradiates a fixed part of the entire migration zone. Although fluorescence is emitted from each migration zone, only fluorescence in a specific lane can pass through the slit 18 and reach the detector. Driver is a slit 19
With this, it is possible to move to the left and right, and it is possible to pass the fluorescence signal from different migration zones at regular intervals. Signals are integrated and data processed in synchronization with the movement of the slit. In this example, detectors may be provided on both sides of the electrophoretic panel, and the slits may be opened and closed in two steps to increase the measurement time per electrophoretic zone.
【００１２】[0012]
【発明の効果】以上説明したように本発明によれば泳動
レーン間隔を光電増倍管などで規定されるよりも小さく
する事ができるため、多くの泳動レーンを必要とする多
種試料の分析に有効である。また、泳動レーン間隔を小
さくする事により温度むらなどによる泳動レーン間の泳
動条件の差を小さくできる利点もある。As described above, according to the present invention, the migration lane interval can be made smaller than that specified by a photomultiplier tube or the like, which is suitable for the analysis of various samples requiring many migration lanes. It is valid. Further, there is an advantage that the difference in the migration condition between migration lanes due to temperature unevenness can be reduced by reducing the migration lane interval.
【図１】本発明の一実施例の平面模式図。FIG. 1 is a schematic plan view of an embodiment of the present invention.
【図２】検出信号の時間変化を示す図であり、（ａ）は
泳動帯毎に整理した蛍光信号の時間変化、（ｂ）は検出
器に入る蛍光信号強度の時間変化、（ｃ）は各泳動帯を
照射する光の時間変化を示す図。FIG. 2 is a diagram showing a time change of a detection signal, (a) a time change of a fluorescence signal arranged for each migration zone, (b) a time change of a fluorescence signal intensity entering a detector, and (c). The figure which shows the time change of the light which irradiates each migration zone.
【図３】本発明の変形例の平面模式図。FIG. 3 is a schematic plan view of a modified example of the present invention.
【図４】本発明によるＤＡＮの分断および泳動分離の様
子を示す説明図。FIG. 4 is an explanatory diagram showing a state of fragmentation and migration separation of DAN according to the present invention.
１…レーザー光、２…部分反射板、３…反射鏡、４…回
転鏡、５…泳動用ガラスパネル、６…泳動ゲル、７…フ
ィルター、８…光電増倍管、９…検出回路、１０…積分
器、１１…同期回路、１２…メモリ、１３…出力機器、
１４’…配列決定しようとするＤＮＡ、１５’…蛍光信
号、１６’…検出器に流れる信号、１７’…断続光パル
ス、１８…可動スリット、１９…ドライバー、２０…光
検出器、２１…ＤＮＡ断片群、２２…泳動方向、２３…
泳動ＤＮＡバンド。DESCRIPTION OF SYMBOLS 1 ... Laser light, 2 ... Partial reflection plate, 3 ... Reflecting mirror, 4 ... Rotating mirror, 5 ... Electrophoresis glass panel, 6 ... Electrophoresis gel, 7 ... Filter, 8 ... Photomultiplier tube, 9 ... Detection circuit, 10 ... integrator, 11 ... synchronization circuit, 12 ... memory, 13 ... output device,
14 '... DNA to be sequenced, 15' ... Fluorescent signal, 16 '... Signal flowing to detector, 17' ... Intermittent light pulse, 18 ... Movable slit, 19 ... Driver, 20 ... Photodetector, 21 ... DNA Fragment group, 22 ... Migration direction, 23 ...
Electrophoretic DNA band.
Claims (7)
り、蛍光体が標識されたＤＮＡ断片が泳動する複数の電
気泳動路と、前記電気泳動路にレーザ光を照射する光照
射手段と、前記レーザ光の照射により前記蛍光体から発
する蛍光を検出する光検出手段とを有し、前記ＤＮＡ断
片を検出するＤＮＡ塩基配列決定装置において、前記光
照射手段は前記レーザ光を走査し、前記複数の電気泳動
路を照射する走査手段を有し、前記光検出手段は、前記
複数の電気泳動路のそれぞれを泳動する前記ＤＮＡ断片
を標識する前記蛍光体からの前記蛍光を、前記レーザ光
の走査に同期して検出することにより、前記電気泳動路
毎に泳動する前記ＤＮＡ断片の前記末端塩基種を識別
し、前記核酸試料の塩基配列を決定することを特徴とす
るＤＮＡ塩基配列決定装置。1. A DNA fragment prepared from a nucleic acid sample, wherein a plurality of electrophoretic paths along which fluorescently labeled DNA fragments migrate, and a light irradiation means for irradiating the electrophoretic path with laser light are provided. A DNA base sequence determination device for detecting the fluorescence emitted from the phosphor upon irradiation of laser light, wherein the light irradiation means scans the laser light, Scanning means for irradiating an electrophoretic path is provided, and the photodetecting means scans the laser light with the fluorescence from the fluorescent substance that labels the DNA fragment that migrates in each of the plurality of electrophoretic paths. DNA base sequence determination, characterized in that the terminal base species of the DNA fragments that migrate in each of the electrophoretic paths are identified by synchronous detection, and the base sequence of the nucleic acid sample is determined. Location.
と前記レーザ光の走査により照射された位置の前記電気
泳動路とを対応させて、前記ＤＮＡ断片の前記末端塩基
種と前記ＤＮＡ断片の長さを求め、前記核酸試料の塩基
配列を決定することを特徴とする請求項１に記載のＤＮ
Ａ塩基配列決定装置。2. The end base species of the DNA fragment and the DNA fragment of the DNA fragment are made to correspond by correlating the time point when the fluorescence is detected by the light detecting means and the electrophoretic path at the position irradiated by the scanning of the laser beam. The DN according to claim 1, wherein the length is determined and the base sequence of the nucleic acid sample is determined.
A base sequencer.
を変化させる光反射手段と、前記レーザ光を互いに異な
る方向に進行する光に分割する光分割手段とを含むこと
を特徴とする請求項１に記載のＤＮＡ塩基配列決定装
置。3. The scanning means includes a light reflecting means for changing the scanning direction of the laser light, and a light splitting means for splitting the laser light into light traveling in mutually different directions. Item 1. A DNA nucleotide sequencer according to item 1.
前記蛍光を検出することを特徴とする請求項１に記載の
ＤＮＡ塩基配列決定装置。4. The DNA base sequencer according to claim 1, wherein the fluorescence is detected by separating each of the electrophoresis paths by time division.
と前記レーザ光の走査により照射された前記電気泳動路
とを対応させ、時分割により前記電気泳動路毎に分離し
て前記蛍光を検出することを特徴とする請求項１に記載
のＤＮＡ塩基配列決定装置。5. The fluorescence is detected by making the time when the fluorescence is detected by the light detecting means and the electrophoretic path irradiated by the scanning of the laser light, and separating the electrophoretic paths by time division. The DNA nucleotide sequencer according to claim 1, wherein
複数の電気泳動路と、前記電気泳動路にレーザ光を照射
する光照射手段と、前記レーザ光の照射により前記蛍光
体から発する蛍光を検出する光検出手段とを有し、前記
ＤＮＡ断片を検出するＤＮＡ塩基配列決定装置におい
て、前記光検出手段は、前記複数の電気泳動路が複数の
群に分けられた各群に対応して設けられた光検出器を含
み、前記各群に対応して設けられた前記光検出器のそれ
ぞれは、前記各群内の前記電気泳動路のそれぞれを泳動
する前記ＤＮＡ断片からの蛍光を時分割により検出する
ことを特徴とするＤＮＡ塩基配列決定装置。6. A plurality of electrophoretic paths along which a DNA fragment labeled with a fluorescent substance migrates, a light irradiating means for irradiating the electrophoretic path with a laser beam, and fluorescence emitted from the fluorescent substance by the irradiation with the laser beam. In the DNA base sequence determination device for detecting the DNA fragment, the light detecting means corresponds to each group in which the plurality of electrophoresis paths are divided into a plurality of groups. Each of the photodetectors provided corresponding to each of the groups includes an provided photodetector, and each of the photodetectors is time-divided from the DNA fragments that migrate in each of the electrophoretic paths in each of the groups. An apparatus for determining a DNA base sequence, which is characterized in that:
複数の電気泳動路と、前記電気泳動路にレーザ光を照射
する光照射手段と、前記レーザ光の照射により前記蛍光
体から発する蛍光を検出する光検出手段とを有し、前記
試料断片を検出するＤＮＡ塩基配列決定装置において、
前記光照射手段は前記レーザ光を走査する走査手段を有
し、前記光検出手段は、前記複数の電気泳動路が複数の
群に分けられた各群に対応して設けられた光検出器を含
み、前記各群に対応して設けられた前記光検出器は、そ
れぞれ前記各群内の前記電気泳動路のそれぞれを泳動す
る前記試料断片からの蛍光を、前記レーザ光の走査に同
期して検出することを特徴とするＤＮＡ塩基配列決定装
置。7. A plurality of electrophoretic paths along which a DNA fragment labeled with a fluorescent substance migrates, a light irradiation means for irradiating the electrophoretic path with a laser beam, and a fluorescence emitted from the fluorescent substance by the irradiation with the laser beam. In a DNA base sequence determination device for detecting the sample fragment, comprising:
The light irradiation means has a scanning means for scanning the laser light, and the light detection means is a photodetector provided corresponding to each group in which the plurality of electrophoresis paths are divided into a plurality of groups. The photodetector provided corresponding to each of the groups, the fluorescence from the sample fragment that migrates in each of the electrophoretic paths in each of the groups, in synchronization with the scanning of the laser light. A DNA nucleotide sequencer characterized by detecting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7305505A JP2679696B2 (en) | 1995-11-24 | 1995-11-24 | DNA base sequencer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7305505A JP2679696B2 (en) | 1995-11-24 | 1995-11-24 | DNA base sequencer |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61245240A Division JP2624655B2 (en) | 1986-10-17 | 1986-10-17 | Electrophoresis device |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9047573A Division JP2776383B2 (en) | 1997-03-03 | 1997-03-03 | DNA base sequencer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH08211017A true JPH08211017A (en) | 1996-08-20 |
| JP2679696B2 JP2679696B2 (en) | 1997-11-19 |
Family
ID=17945971
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7305505A Expired - Lifetime JP2679696B2 (en) | 1995-11-24 | 1995-11-24 | DNA base sequencer |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2679696B2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57204437A (en) * | 1981-06-11 | 1982-12-15 | Hiranuma Sangyo Kk | Measuring method for interval of inspection body in concentration measuring apparatus |
| JPS6162843A (en) * | 1984-08-13 | 1986-03-31 | Hitachi Ltd | Fluorescence detection type electrophoretic apparatus |
-
1995
- 1995-11-24 JP JP7305505A patent/JP2679696B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57204437A (en) * | 1981-06-11 | 1982-12-15 | Hiranuma Sangyo Kk | Measuring method for interval of inspection body in concentration measuring apparatus |
| JPS6162843A (en) * | 1984-08-13 | 1986-03-31 | Hitachi Ltd | Fluorescence detection type electrophoretic apparatus |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2679696B2 (en) | 1997-11-19 |
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