JP2648082B2 - Nucleic acid base detection method - Google Patents
Nucleic acid base detection methodInfo
- Publication number
- JP2648082B2 JP2648082B2 JP5201479A JP20147993A JP2648082B2 JP 2648082 B2 JP2648082 B2 JP 2648082B2 JP 5201479 A JP5201479 A JP 5201479A JP 20147993 A JP20147993 A JP 20147993A JP 2648082 B2 JP2648082 B2 JP 2648082B2
- Authority
- JP
- Japan
- Prior art keywords
- nucleic acid
- detecting
- labeled
- acid fragment
- molecular weight
- 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 - Fee Related
Links
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- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は核酸塩基検出方法にかか
わり、特に、核酸検出法の装置化に際し、その高精度化
に好適な核酸塩基検出方法に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a nucleic acid base, and more particularly to a method for detecting a nucleic acid base which is suitable for improving the accuracy of a nucleic acid detecting method .
【０００２】[0002]
【従来の技術】従来の核酸検出法は、以下の２法のうち
いずれかを用いていたが、いずれにおいても下記の欠点
が存在した。すなわち、一つは、32Ｐ、35Ｓなどの放射
性同位体により標識した核酸を、Ｘ線フィルム、Ｇ．
Ｍ．管、シンチレーションカウンタ等の放射線検出器で
検出する方法で、この方法は高感度（＜０．１ｐｇ）で
はあるが、安全性上、法規上の規制があり、取扱いに不
便があった。一方、エチジウム・ブロミド、アクリジン
・オレンジ、プロフラビン等の蛍光色素で標識した核酸
に紫外線を照射し、蛍光を発生させて、これを検出する
方法（例えば、蛋白質・核酸・酵素 別冊：蛍光測定の
原理と生体系への応用、ｐｐ．２０６−２３１）は、安
全ではあるが、感度に乏しく（前者の方法の場合の１／
１００以下）、核酸が極微量（＜１ｎｇ）である場合な
どには、実用的でなかった。2. Description of the Related Art Conventional nucleic acid detection methods employ either one of the following two methods, but both have the following disadvantages. That is, first, a nucleic acid labeled with a radioactive isotope such as 32 P, 35 S is applied to an X-ray film, G.
M. This is a method of detecting with a radiation detector such as a tube or a scintillation counter. Although this method has high sensitivity (<0.1 pg), it is inconvenient to handle due to safety regulations and regulations. On the other hand, a nucleic acid labeled with a fluorescent dye such as ethidium bromide, acridine orange, or proflavine is irradiated with ultraviolet light to generate fluorescence, and a method for detecting the fluorescence (for example, Protein, Nucleic Acid, Enzyme Separate Volume: The principle and application to biological systems, pp. 206-231) are safe, but have poor sensitivity (1/1 of the former method).
100 or less), and was not practical when the amount of nucleic acid was very small (<1 ng).
【０００３】[0003]
【発明が解決しようとする課題】上記従来技術は、放射
性同位体で標識した核酸を放射線検出器で検出する方法
では安全性に問題があり、蛍光色素で標識した核酸を紫
外線を用いて検出する方法では感度に乏しいという問題
があった。In the above-mentioned prior art, there is a problem in safety in the method of detecting a nucleic acid labeled with a radioisotope with a radiation detector, and the nucleic acid labeled with a fluorescent dye is detected using ultraviolet rays. The method has a problem that the sensitivity is poor.
【０００４】本発明の目的は、高感度（〜１ｐｇ）でか
つ安全性の高い核酸塩基検出方法を提供することにあ
る。It is an object of the present invention to provide a highly sensitive (up to 1 pg) and highly safe method for detecting nucleobases .
【０００５】[0005]
【課題を解決するための手段】本発明は、検出すべき核
酸または核酸混合物に、あらかじめ、天然の核酸中には
含まれていないかまたは含有量が極めて少なく、かつそ
の導入によって核酸全体の化学的性質を大きく変化させ
ない特定の元素でもって標識しておき、分子量分離した
核酸断片中の該特定の元素を同定し、これによって核酸
断片を検出するのが特徴である。SUMMARY OF THE INVENTION The present invention relates to a nucleic acid or a nucleic acid mixture to be detected which is not contained in a natural nucleic acid or has a very low content, and which is introduced into the nucleic acid or the nucleic acid mixture. The method is characterized by labeling with a specific element that does not significantly change its physical properties, identifying the specific element in the nucleic acid fragment separated from the molecular weight, and detecting the nucleic acid fragment.
【０００６】上記特定の元素としては、例えばＳ、Ｂ
ｒ、Ｉのような非金属元素や、Ａｇ、Ａｕ、Ｐｔ、Ｏ
ｓ、Ｈｇのような金属元素がある。The specific elements include, for example, S, B
non-metallic elements such as r, I, Ag, Au, Pt, O
There are metal elements such as s and Hg.
【０００７】また、これら特定の元素を同定する方法と
して、原子吸光分析法、またはプラズマ発光分析法を用
いる。As a method for identifying these specific elements, an atomic absorption analysis method or a plasma emission analysis method is used.
【０００８】[0008]
【作用】本発明によれば、このような特定の元素を核酸
または核酸混合物に導入しておくことにより、該核酸に
対して種々の化学的操作を施した後でも、これを分離
し、各成分を抽出し、特定標識元素を同定することで、
被標識核酸成分を高感度でかつ安全に検出することがで
きる。According to the present invention, by introducing such a specific element into a nucleic acid or a nucleic acid mixture, the nucleic acid can be separated even after various chemical operations are performed on the nucleic acid. By extracting components and identifying specific labeling elements,
The nucleic acid component to be labeled can be detected with high sensitivity and safely.
【０００９】[0009]
【実施例】以下、本発明の実施例を図１ないし図３によ
って説明する。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIGS.
【００１０】本実施例では、核酸試料は、あらかじめ、
天然の核酸中には含まれていないか、またはその含有量
が極めて少ない元素で標識しておく。例えば、図１に示
すように、正常基質の代りに、リン酸エステル結合部位
の酸素元素をイオウ原子に置換したヌクレオチド（ジデ
オキシアデノシン−３−〔α−Ｓ〕リン酸）をデオキシ
リポ核酸（ＤＮＡ）断片の末端に生化学的に結合してお
く。In this embodiment, the nucleic acid sample is prepared in advance.
The nucleic acid is labeled with an element that is not contained in the natural nucleic acid or has an extremely small content. For example, as shown in FIG. 1, instead of a normal substrate, a nucleotide (dideoxyadenosine-3- [α-S] phosphate) in which the oxygen element in the phosphate ester binding site is replaced with a sulfur atom is deoxyliponucleic acid (DNA). Biochemically bind to the ends of the fragments.
【００１１】図２は、電気泳動法により分子量分離をす
る場合に本発明を適用した一実施例で用いる装置全体を
示したものである。装置は、試料溶液槽１、送液ポンプ
２、電気泳動用緩衝液槽３、泳動分離用アクリルアミド
ゲル（以下ゲルと記す）４、標識元素検出部９、および
泳動分離帯溶出用緩衝液槽１２を主な構成要素とする。FIG. 2 shows an entire apparatus used in an embodiment to which the present invention is applied when molecular weight is separated by an electrophoresis method. The apparatus includes a sample solution tank 1, a liquid sending pump 2, an electrophoresis buffer solution tank 3, an acrylamide gel for electrophoresis separation (hereinafter, referred to as gel) 4, a labeling element detection unit 9, and a buffer solution tank 12 for elution of a migration separation zone. Is the main component.
【００１２】次に、検出の手順を説明する。試料溶液槽
１からの標識された核酸試料を送液ポンプ２によりゲル
４の負極側にのせ、ゲル４の両端が電気泳動用緩衝液槽
３に接するようになし、高圧直流電源１３による５０Ｖ
／ｃｍ程度の電圧で泳動させる。すると、同一分子量を
有する核酸成分はそれぞれ泳動分離帯６を形成しつつ負
極から正極に向い、分子量の対数にほぼ反比例した移動
度で泳動する。一方、ゲル４の正極端よりも幾分か負極
端側に寄った付近の電気絶縁板５に設けた窓７を通し
て、泳動分離帯溶出用緩衝液槽１２からの、ゲル内液と
同一組成の緩衝液をゆっくりと流すとともに、この緩衝
液に、低圧直流電源１１により、泳動方向の電位勾配を
大きくは乱さない程度の電圧をかける。その結果、分離
された泳動体は溶出した泳動分離帯８として回収され、
標識元素検出部９に送られる。標識元素検出部９では、
例えば、金属元素に対しては原子吸光分析装置（感度〜
ｐｐｔ）、プラズマ発光分析装置（感度〜ｐｐｂ）によ
り、ヨウ素や本実施例で用いたイオウに対しては、酸化
して二酸化イオウ（イオウの場合）とした後、質量分析
装置（感度＜ｐｐｂ）により、それぞれ標識元素を同定
できるので、分子量の小さいものから順に溶出してくる
被標識核酸断片を検出することができる。Next, the detection procedure will be described. The labeled nucleic acid sample from the sample solution tank 1 is placed on the negative electrode side of the gel 4 by the liquid sending pump 2 so that both ends of the gel 4 are in contact with the electrophoresis buffer solution tank 3.
Electrophoresis at a voltage of about / cm. Then, the nucleic acid components having the same molecular weight move from the negative electrode to the positive electrode while forming the electrophoretic separation zone 6, and migrate with a mobility substantially inversely proportional to the logarithm of the molecular weight. On the other hand, through the window 7 provided in the electric insulating plate 5 near the negative electrode end somewhat from the positive electrode end of the gel 4, the same composition as the gel internal solution from the electrophoresis separation band elution buffer tank 12 was passed. The buffer is slowly flowed, and a voltage is applied to the buffer by the low-voltage DC power supply 11 to such an extent that the potential gradient in the migration direction is not greatly disturbed. As a result, the separated electrophores are collected as eluted electrophoretic separation bands 8,
It is sent to the labeling element detecting section 9. In the labeling element detector 9,
For example, an atomic absorption spectrometer (sensitivity ~
ppt), iodine and sulfur used in this example are oxidized to sulfur dioxide (in the case of sulfur) by a plasma emission analyzer (sensitivity to ppb), and then mass spectrometer (sensitivity <ppb). Thus, the labeling element can be identified, whereby the nucleic acid fragment to be labeled eluted in order from the one with the smallest molecular weight can be detected.
【００１３】図３は、高速ゲル濾過法により分子量分離
をする場合に本発明を適用した他の実施例で用いる装置
全体を示したものである。装置は、溶離液槽１５、送液
ポンプ２、試料溶液槽１、高速ゲル濾過剤である充填剤
１４、および標識元素検出部９を主な構成要素とする。
検出の手順は、試料溶液槽１中の標識された核酸溶液
を、溶離液槽１５中の溶離液と共に、送液ポンプ２によ
り充填剤１４中に移送すると、充填剤１４のもつ分子ふ
るい効果または分配効果によって、核酸断片は質量数の
小さいものから順に分離帯１６を形成し、これらは標識
元素検出部９側に移動する。これら分離帯１６は、標識
元素検出部９において、前の実施例で述べたと同様の方
法により、連続的に検出できる。FIG. 3 shows an entire apparatus used in another embodiment to which the present invention is applied when molecular weight is separated by high-speed gel filtration . The main components of the apparatus are an eluent tank 15, a liquid sending pump 2, a sample solution tank 1, a filler 14, which is a high-speed gel filtration agent , and a labeling element detection unit 9.
The detection procedure is as follows. When the labeled nucleic acid solution in the sample solution tank 1 is transferred into the packing material 14 by the liquid sending pump 2 together with the eluate in the eluent tank 15, the molecular sieving effect of the packing material 14 or Due to the distribution effect, the nucleic acid fragments form separation bands 16 in ascending order of mass number, and these migrate to the labeling element detection unit 9 side. These separation zones 16 can be continuously detected by the labeling element detecting section 9 by the same method as described in the previous embodiment.
【００１４】[0014]
【発明の効果】本発明によれば、従来の核酸検出方法に
比べて、次のような効果が得られる。According to the present invention, the following effects can be obtained as compared with the conventional nucleic acid detection method .
【００１５】（イ）核酸の化学的、生化学的性質をほと
んど変化させることなく、かつ安全性の高い標識物が使
用でき、安全で高感度の検出ができる。(A) A highly safe label can be used with little change in the chemical and biochemical properties of the nucleic acid, and safe and highly sensitive detection can be performed.
【００１６】（ロ）分子量分離操作はフロー型で行うの
で、従来の電気泳動法に比し、測定時間の短縮、測定試
料の微量化が図れる。(B) Since the molecular weight separation operation is performed in a flow type, the measurement time can be reduced and the amount of the sample to be measured can be reduced as compared with the conventional electrophoresis method.
【図１】本発明により特定の元素で標識する例を示す説
明図である。FIG. 1 is an explanatory diagram showing an example of labeling with a specific element according to the present invention.
【図２】本発明の一実施例で用いる装置の構成を示す構
成図である。FIG. 2 is a configuration diagram showing a configuration of an apparatus used in one embodiment of the present invention.
【図３】本発明の他の実施例で用いる装置の構成を示す
構成図である。FIG. 3 is a configuration diagram showing a configuration of an apparatus used in another embodiment of the present invention.
１ 試料溶液槽 ２ 送液ポンプ ３ 電気泳動用緩衝液槽 ４ 泳動分離用アクリルアミドゲル ５ 電気絶縁板 ６ 泳動分離帯 ７ 窓 ８ 溶出した泳動分離帯 ９ 標識元素検出部 １１ 低圧直流電源 １２ 泳動分離帯溶出用緩衝液槽 １３ 高圧直流電源 １４ 充填剤 １５ 溶離液槽 １６ 分離帯 REFERENCE SIGNS LIST 1 sample solution tank 2 liquid sending pump 3 buffer buffer for electrophoresis 4 acrylamide gel for electrophoretic separation 5 electrical insulating plate 6 electrophoretic separation band 7 window 8 eluted electrophoretic separation band 9 labeling element detection unit 11 low-voltage DC power supply 12 electrophoretic separation band Elution buffer tank 13 High voltage DC power supply 14 Filler 15 Eluent tank 16 Separation zone
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｎ 15/00 9282−4Ｂ Ｃ１２Ｎ 15/00 (72)発明者 鴇田 二郎 東京都国分寺市東恋ヶ窪１丁目280番地 株式会社日立製作所 中央研究所内 (72)発明者 永井 啓一 東京都国分寺市東恋ヶ窪１丁目280番地 株式会社日立製作所 中央研究所内 (72)発明者 岡田 修身 東京都国分寺市東恋ヶ窪１丁目280番地 株式会社日立製作所 中央研究所内──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. 6 Identification number Agency reference number FI Technical indication location C12N 15/00 9282-4B C12N 15/00 (72) Inventor Jiro Tokita 1-chome Higashi-Koigabo, Kokubunji-shi, Tokyo 280 Hitachi Central Research Laboratory Co., Ltd. Hitachi Central Research Laboratory
Claims (1)
は高速ゲル濾過法を用いて分子量分離することにより核
酸断片を検出する核酸塩基検出方法であって、前記核酸
断片は天然の核酸中には含まれていないかまたは含有量
が極めて少なく、かつその導入によって核酸全体の化学
的性質を大きく変化させない特定の元素で標識されてお
り、前記分子量分離して得た核酸断片中の前記特定の元
素を、原子吸光分析法、またはプラズマ発光分析法で同
定することにより核酸断片を検出することを特徴とする
核酸塩基検出方法。1. The method according to claim 1, wherein the labeled nucleic acid fragment is subjected to electrophoresis,
Is a nucleic acid base detection method for detecting a nucleic acid fragment by separating the molecular weight using a high-speed gel filtration method , wherein the nucleic acid fragment is not contained in natural nucleic acid or has a very low content, and The nucleic acid is labeled with a specific element that does not significantly change the chemical properties of the entire nucleic acid upon introduction, and the specific element in the nucleic acid fragment obtained by the molecular weight separation is identified by atomic absorption analysis or plasma emission analysis. A method for detecting a nucleic acid base by detecting a nucleic acid fragment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5201479A JP2648082B2 (en) | 1984-06-28 | 1993-08-13 | Nucleic acid base detection method |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59131909A JPH0658368B2 (en) | 1984-06-28 | 1984-06-28 | Nucleic acid base detector |
| JP5201479A JP2648082B2 (en) | 1984-06-28 | 1993-08-13 | Nucleic acid base detection method |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59131909A Division JPH0658368B2 (en) | 1984-06-28 | 1984-06-28 | Nucleic acid base detector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0666791A JPH0666791A (en) | 1994-03-11 |
| JP2648082B2 true JP2648082B2 (en) | 1997-08-27 |
Family
ID=26466619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5201479A Expired - Fee Related JP2648082B2 (en) | 1984-06-28 | 1993-08-13 | Nucleic acid base detection method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2648082B2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0063879A2 (en) * | 1981-04-17 | 1982-11-03 | Yale University | Modified nucleotides and methods of preparing and using same |
| JPS59100856A (en) * | 1982-12-01 | 1984-06-11 | Hitachi Ltd | Analysis device connecting liquid chromatograph and mass spectrometer |
-
1993
- 1993-08-13 JP JP5201479A patent/JP2648082B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0063879A2 (en) * | 1981-04-17 | 1982-11-03 | Yale University | Modified nucleotides and methods of preparing and using same |
| JPS59100856A (en) * | 1982-12-01 | 1984-06-11 | Hitachi Ltd | Analysis device connecting liquid chromatograph and mass spectrometer |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0666791A (en) | 1994-03-11 |
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