JPH0574780B2 - - Google Patents
Info
- Publication number
- JPH0574780B2 JPH0574780B2 JP59167813A JP16781384A JPH0574780B2 JP H0574780 B2 JPH0574780 B2 JP H0574780B2 JP 59167813 A JP59167813 A JP 59167813A JP 16781384 A JP16781384 A JP 16781384A JP H0574780 B2 JPH0574780 B2 JP H0574780B2
- Authority
- JP
- Japan
- Prior art keywords
- gel
- light
- light irradiation
- gel electrophoresis
- electrophoresis device
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000001502 gel electrophoresis Methods 0.000 claims description 17
- 238000001514 detection method Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 230000003287 optical effect Effects 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 230000005684 electric field Effects 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 3
- 238000013508 migration Methods 0.000 claims description 3
- 230000005012 migration Effects 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims 3
- 239000012634 fragment Substances 0.000 description 11
- 238000001962 electrophoresis Methods 0.000 description 8
- 239000011521 glass Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000003234 fluorescent labeling method Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000007740 vapor deposition Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
Description
【発明の詳細な説明】
〔発明の利用分野〕
本発明はDNA，RNAなどの塩基配列決定用の
ゲル電気泳動装置の改良に関し、とくにDNAな
どのフラグメント光を実時間で検出できるように
改良されたゲル電気泳動装置に関するものであ
る。[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to an improvement of a gel electrophoresis device for determining base sequences of DNA, RNA, etc., and in particular, to an improvement of a gel electrophoresis device for determining DNA, RNA, etc. fragment light in real time. The present invention relates to a gel electrophoresis device.
DNAやRNA等の塩基配列決定にはラジオアイ
ソトープラベル法が通常用いられている。しか
し、使用上の不便さから蛍光体ラベル法などが次
世代技術として開発途上にある（昭和58年度科学
研究費補助金研究成果報告集、S59年３月、pp20
−25）。この蛍光体ラベル法ではDNA等のフラグ
メントの一部に蛍光体を付加して泳動させ、レー
ザー光などで照射することによつて発生する蛍光
を観測するものである。現在、この手法の難点の
１つはレーザ光などで光励起すると、電気泳動ゲ
ル自体からも蛍光や散乱光が出て、これがフラグ
メント光のバツクグラウンドとなり、測定限界を
決めてしまうため、計測の信号対雑音比（Ｓ／Ｎ
比）が上がらない事であつた。
Radioisotope labeling is commonly used to determine base sequences of DNA, RNA, etc. However, due to the inconvenience of use, phosphor labeling methods are currently under development as next-generation technologies (Report on Research Results of Grants-in-Aid for Scientific Research 1985, March 1983, pp. 20
−25). In this fluorophore labeling method, a fluorophore is attached to a portion of a fragment of DNA or the like, the fragment is caused to migrate, and the fluorescence generated by irradiation with laser light or the like is observed. Currently, one of the difficulties with this method is that when photoexcited with laser light, etc., fluorescence and scattered light are emitted from the electrophoretic gel itself, which becomes the background of fragment light and determines the measurement limit, so the measurement signal cannot be measured. Noise-to-noise ratio (S/N
The problem was that the ratio) did not increase.
本発明の目的は蛍光体ラベル法によるDNA等
のフラグメント光の検出に用いらるゲル電気泳動
装置において、電気泳動ゲルから発する蛍光ある
いは散乱光を排除し、実時間光検出方式による
DNA等の塩基配列決定を可能とすることである。
The purpose of the present invention is to eliminate fluorescence or scattered light emitted from the electrophoresis gel in a gel electrophoresis device used for detecting fragment light of DNA etc. using the fluorescent labeling method, and to use a real-time optical detection method.
The objective is to enable base sequencing of DNA, etc.
上記目的を達成するために、本発明では、ゲル
電気泳動装置における光照射部（または蛍光取出
部）にはバツクグランドとなる蛍光や散乱光を発
するものが存在しないような構造とし、バツクグ
ランドノイズを大巾に低減させた。すなわち、本
発明では、ゲル電気泳動路中にゲルを含まないか
あるいはこのゲルとは組成の異なる物質からなる
部分領域を設けてなることを特徴としている。
In order to achieve the above object, in the present invention, the light irradiation part (or fluorescence extraction part) in the gel electrophoresis apparatus is structured so that there is no background that emits fluorescence or scattered light, and background noise is eliminated. has been drastically reduced. That is, the present invention is characterized in that the gel electrophoresis path is provided with a partial region that does not contain gel or is made of a substance having a composition different from that of the gel.
以下本発明の一実施例を第１図を参照して説明
する。第１図は本発明の一実施例になるゲル電気
泳動装置の概略構成を示しており、図中の１は無
蛍光ガラスからなるゲル保持板で、２はバツフア
ー液、３は電気泳動路を形成しているゲル、４は
本発明による無ゲル領域を示している。５は入射
光制限スリツト、６は蛍光制限スリツト、７は集
光レンズ、８は干渉フイルター、９は光検出器、
１０は記録計、１５はレーザー等の紫外光源であ
る。この光源１５により、ゲル電気泳動路に、紫
外光をあてた時、蛍光を発するのは重合した（約
５〜20％濃度のポリアクリルアミド）ゲルの部分
が主である。そこで本発明では泳動ゲル３によつ
て形成される電気泳動路の中間にゲルのない領域
４を作り、ここに光源１５からの紫外線１５′を
照射するようにしてある。この領域４にはバツフ
アー溶液（たとえばTris−BoroteEDTAバツフ
アー）が満たされているが紫外線を照射しても蛍
光は出さない。また紫外線１５′はこの無ゲル領
域４にだけ当たるようにスリツト５により照射領
域が制限されている。蛍光体で標識されたDNA
フラグメントがゲル中を泳動してこの領域４に到
達すると紫外線１５′で照射され、蛍光を発する。
この蛍光はレンズ７等で集光され、フイルター８
を通過した後に光検出器９に入る。励起用の紫外
光源１５としてはレーザー等が用いられるが、入
射光強度は蛍光に比べて非常に強く散乱光などが
無ゲル領域４との境界近傍にあるゲルにあたつて
発せられる蛍光に起因するバツクグランドノイズ
を除去するために受光側にも第１図に示したよう
にスリツト６を設けている。また、ゲル保持板１
は通常のガラスでは蛍光を出すので、この実施例
では無蛍光ガラスが使用されている。第１図の実
施例ではレーザー光１５′はゲル保持板１を通し
て照射部４に入るが、第２図はレーザー光１５′
を横方向からゲル保持板を介さずに入射させた例
である。この例では両側のガラス板１により入射
光１５′はほぼ全反射されるので一種のオプチカ
ルガイドが形成される。また、この例では反射板
１２を用いてレーザー光が照射部を何度も通過で
きるように工夫してある。また、DNAフラグメ
ントからの蛍光は反射板１３によつて反射され
る。この結果感度が増大する。この方式では照射
領域の巾（図の垂直方向）が長いので十分平行な
レーザー光を用いる必要がある。この場合はガラ
ス板１からの蛍光はほとど出ない利点がある。
An embodiment of the present invention will be described below with reference to FIG. FIG. 1 shows a schematic configuration of a gel electrophoresis apparatus according to an embodiment of the present invention. In the figure, 1 is a gel holding plate made of non-fluorescent glass, 2 is a buffer liquid, and 3 is an electrophoresis path. The formed gel, 4, indicates the gel-free area according to the present invention. 5 is an incident light limiting slit, 6 is a fluorescence limiting slit, 7 is a condensing lens, 8 is an interference filter, 9 is a photodetector,
10 is a recorder, and 15 is an ultraviolet light source such as a laser. When ultraviolet light is applied to the gel electrophoresis channel by this light source 15, it is mainly the polymerized gel (polyacrylamide with a concentration of about 5 to 20%) that emits fluorescence. Therefore, in the present invention, a gel-free area 4 is created in the middle of the electrophoresis path formed by the electrophoresis gel 3, and the ultraviolet ray 15' from the light source 15 is irradiated to this area. Although this region 4 is filled with a buffer solution (for example, Tris-Borote EDTA buffer), no fluorescence is emitted even when irradiated with ultraviolet rays. Further, the irradiation area of the ultraviolet rays 15' is limited by the slits 5 so that the ultraviolet rays 15' only hit this gel-free area 4. DNA labeled with fluorophore
When the fragments migrate through the gel and reach this region 4, they are irradiated with ultraviolet light 15' and emit fluorescence.
This fluorescence is collected by lens 7, etc., and passed through filter 8.
After passing through, it enters the photodetector 9. A laser or the like is used as the ultraviolet light source 15 for excitation, but the intensity of the incident light is much stronger than that of fluorescence, and the scattered light is caused by the fluorescence emitted when the scattered light hits the gel near the boundary with the gel-free region 4. In order to remove the background noise caused by this, a slit 6 is provided on the light receiving side as shown in FIG. In addition, gel retaining plate 1
Since ordinary glass emits fluorescence, non-fluorescent glass is used in this embodiment. In the embodiment shown in FIG. 1, the laser beam 15' enters the irradiation section 4 through the gel holding plate 1, but in the embodiment shown in FIG.
This is an example in which the light is incident from the lateral direction without passing through the gel holding plate. In this example, the incident light 15' is almost totally reflected by the glass plates 1 on both sides, so that a kind of optical guide is formed. Further, in this example, a reflection plate 12 is used so that the laser beam can pass through the irradiation section many times. Further, the fluorescence from the DNA fragment is reflected by the reflecting plate 13. This results in increased sensitivity. In this method, since the width of the irradiation area (in the vertical direction in the figure) is long, it is necessary to use sufficiently parallel laser beams. In this case, there is an advantage that almost no fluorescence is emitted from the glass plate 1.
通常の電気泳動装置では泳動部の両端に直流電
圧をかける。DNAフラグメントの泳動速度は電
界強度、ゲル重合度、その他に依存する。上記の
無ゲル領域４で泳動速度が早くなり、蛍光検出の
時間が十分に得らない事がある。そこで本実施例
では第３図に示すように電極１４間に加える電位
を変化させることにより無ゲル領域での電界強度
を弱くし、DNAフラグメントの光照射領域４に
おける滞在時間を長くしている。 In a normal electrophoresis device, a DC voltage is applied to both ends of the electrophoresis section. The migration speed of DNA fragments depends on electric field strength, gel polymerization degree, and other factors. In the above-mentioned gel-free region 4, the electrophoresis speed becomes high, and sufficient time for fluorescence detection may not be obtained. Therefore, in this embodiment, as shown in FIG. 3, by changing the potential applied between the electrodes 14, the electric field strength in the gel-free region is weakened, and the residence time of the DNA fragments in the light irradiation region 4 is lengthened.
以上説明したように本発明によば、蛍光体ラベ
ルされたDNA等のフラグメント以外の物質から
蛍光が出ることを防止することができる。この結
果、泳動してくるDNA等のフラグメントを光を
用いて直接検出する際、従来法に比べて約１桁の
Ｓ／Ｎ向上が計れる。
As explained above, according to the present invention, it is possible to prevent fluorescence from being emitted from substances other than fluorescent substance-labeled fragments such as DNA. As a result, when directly detecting migrating fragments of DNA or the like using light, it is possible to improve the S/N by about one order of magnitude compared to conventional methods.
上記技術を用いることによりこれまでＳ／Ｎが
悪いため実用に到らなかつた光直接検出方式を用
いたDNA塩基配列決定が可能となる。 By using the above technology, it becomes possible to determine the DNA base sequence using a direct optical detection method, which has hitherto been impractical due to poor S/N ratio.
第１図は装置の縦断面図である。第２図は無ゲ
ル領域で切断した時の泳動装置の横断面図であ
る。第３図は無ゲル領域近傍の拡大縦断面図であ
る。
１……無蛍光ガラス製ゲル保持板、２……バツ
フアー液、３……ゲル、４……無ゲル領域、５…
…入射光制限スリツト、６……蛍光制限スリト、
７……集光レンズ、８……干渉フイルター、９…
…光検出器、１０……記録計、１１……DNAバ
ンド、１２……反射ミラー、１３……反射板、１
４……電界強度コントロール用蒸着電極、１５…
…レーザー光源、１６……レンズ、１７……スリ
ト、１８……集光レンズ、１９……検出器。
FIG. 1 is a longitudinal sectional view of the device. FIG. 2 is a cross-sectional view of the electrophoresis device when cut in a gel-free region. FIG. 3 is an enlarged vertical cross-sectional view of the vicinity of the gel-free region. 1... Gel holding plate made of non-fluorescent glass, 2... Buffer liquid, 3... Gel, 4... Gel-free area, 5...
... Incident light limiting slit, 6... Fluorescence limiting slit,
7...Condensing lens, 8...Interference filter, 9...
...Photodetector, 10...Recorder, 11...DNA band, 12...Reflection mirror, 13...Reflection plate, 1
4... Vapor deposition electrode for electric field strength control, 15...
...Laser light source, 16...Lens, 17...Slit, 18...Condensing lens, 19...Detector.
Claims (1)
状に配置されたゲルと、前記試料に泳動力を付与
するための電界を発生する手段とを具備するゲル
電気泳動装置において、前記ゲルとは組成の異な
る物質からなる光照射領域が、前記試料の泳動す
る方向とほぼ直交する方向に、前記ゲルに連接し
て配置され、前記蛍光体を励起する励起光を発生
し励起光を前記光照射領域に照射するための光照
射手段と、前記光照射領域に泳動してくる前記試
料に標識された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することを特徴とするゲル
電気泳動装置。 ２ 前記ゲルのなす面の一方の外側に前記光照射
手段が配置され、前記ゲルのなす面の他方の外側
に前記光検出手段が配置されたことを縛徴とする
特許請求の範囲第１項に記載のゲル電気泳動装
置。 ３ 実質的に前記光照射領域だけに前記励起光を
照射するための第１の光学的スリツトが前記ゲル
のなす面の一方の外側に設けら、前記光照射領域
で前記蛍光体から発する蛍光だけを実質的に取り
出すための第２の光学的スリツトが前記ゲルのな
す面の他方の外側に設けられたことを特徴とする
特許請求の範囲第２項に記載のゲル電気泳動装
置。 ４ 前記試料の泳動方向とほぼ直交し前記ゲルの
なす面と平行な一方の方向から前記励起光を前記
光照射領域に照射する前記光照射手段が設けら
れ、前記ゲルのなす面の外側に前記光検出手段が
配置されたことを特徴とする特許請求の範囲第１
項に記載のゲル電気泳動装置。 ５ 前記試料の泳動方向とほぼ直交し前記ゲルの
なす面と平行な他方の方向の前記光照射領域の外
側に、前記励起光を反射するための反射ミラーが
設けられたことを特徴とする特許請求の範囲第４
項に記載のゲル電気泳動装置。 ６ 前記ゲルのなす面の他方の外側に前記光照射
領域と対向して反射板が設けらたことを特徴とす
る特許請求の範囲第４項または第５項に記載のゲ
ル電気泳動装置。 ７ 前記光照射領域がバツフアー溶液からなるこ
とを特徴とする特許請求の範囲第１項から第６項
のいずかに記載のゲル電気泳動装置。 ８ 前記起光がレーザ光であることを特徴とする
特許請求の範囲第１項から第７項のいずかに記載
のゲル電気泳動装置。[Scope of Claims] 1. A gel electrophoresis apparatus comprising a planar gel on which a fluorescently labeled sample migrates, and means for generating an electric field to impart electrophoretic force to the sample. , a light irradiation area made of a substance having a different composition from the gel is arranged in connection with the gel in a direction substantially perpendicular to the direction in which the sample migrates, and generates excitation light to excite the phosphor. It is characterized by comprising a light irradiation means for irradiating the light irradiation area with light, and a light detection means for detecting fluorescence emitted from the fluorescent substance labeled on the sample migrating to the light irradiation area. Gel electrophoresis device. 2. Claim 1, characterized in that the light irradiation means is arranged outside one of the surfaces formed by the gel, and the light detection means is arranged outside the other surface formed by the gel. The gel electrophoresis device described in . 3. A first optical slit for irradiating the excitation light substantially only to the light irradiation area is provided on the outside of one of the surfaces of the gel, and only the fluorescence emitted from the phosphor in the light irradiation area is provided. 3. The gel electrophoresis device according to claim 2, wherein a second optical slit for substantially taking out the gel is provided on the outside of the other surface formed by the gel. 4. The light irradiation means is provided for irradiating the light irradiation region with the excitation light from one direction substantially perpendicular to the migration direction of the sample and parallel to the surface formed by the gel, and Claim 1 characterized in that a light detection means is arranged.
The gel electrophoresis device described in Section 1. 5. A patent characterized in that a reflecting mirror for reflecting the excitation light is provided outside the light irradiation area in the other direction substantially perpendicular to the migration direction of the sample and parallel to the plane formed by the gel. Claim No. 4
The gel electrophoresis device described in Section 1. 6. The gel electrophoresis device according to claim 4 or 5, characterized in that a reflecting plate is provided on the other outside of the surface formed by the gel, facing the light irradiation area. 7. The gel electrophoresis device according to any one of claims 1 to 6, wherein the light irradiation area is made of a buffer solution. 8. The gel electrophoresis device according to any one of claims 1 to 7, wherein the light emission is a laser beam.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59167813A JPS6147549A (en) | 1984-08-13 | 1984-08-13 | Gel electrophoretic apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59167813A JPS6147549A (en) | 1984-08-13 | 1984-08-13 | Gel electrophoretic apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6147549A JPS6147549A (en) | 1986-03-08 |
| JPH0574780B2 true JPH0574780B2 (en) | 1993-10-19 |
Family
ID=15856578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59167813A Granted JPS6147549A (en) | 1984-08-13 | 1984-08-13 | Gel electrophoretic apparatus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6147549A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4810348A (en) * | 1987-03-16 | 1989-03-07 | Helena Laboratories Corporation | Automatic electrophoresis apparatus and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5028397A (en) * | 1973-04-05 | 1975-03-22 | ||
| JPS5243598A (en) * | 1975-10-01 | 1977-04-05 | Max Co Ltd | Rope-binding device |
| JPS5937557B2 (en) * | 1979-10-02 | 1984-09-10 | 株式会社 日立メディコ | X-ray tube filament heating device |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5937557U (en) * | 1982-09-01 | 1984-03-09 | 株式会社島津製作所 | Capillary gel isotachophoresis analyzer |
-
1984
- 1984-08-13 JP JP59167813A patent/JPS6147549A/en active Granted
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5028397A (en) * | 1973-04-05 | 1975-03-22 | ||
| JPS5243598A (en) * | 1975-10-01 | 1977-04-05 | Max Co Ltd | Rope-binding device |
| JPS5937557B2 (en) * | 1979-10-02 | 1984-09-10 | 株式会社 日立メディコ | X-ray tube filament heating device |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6147549A (en) | 1986-03-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |