JPS613063A - Measurement of polynucleotide utilizing luminous substance - Google Patents
Measurement of polynucleotide utilizing luminous substanceInfo
- Publication number
- JPS613063A JPS613063A JP59123757A JP12375784A JPS613063A JP S613063 A JPS613063 A JP S613063A JP 59123757 A JP59123757 A JP 59123757A JP 12375784 A JP12375784 A JP 12375784A JP S613063 A JPS613063 A JP S613063A
- Authority
- JP
- Japan
- Prior art keywords
- substance
- polynucleotide
- luminescent substance
- measured
- luminous
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
特定の構造を有するデオキシリ−核酸（ＤＮＡ）及びＩ
Ｊ ｇ核酸（ＲＮＡ）の測定は生化学分野において重要
であり、例えば人血清中のＤＮＡを測定することによっ
てウィルス感染の検査あるいは遺伝性疾患の発見などを
行なうことができる。本発明はこのようなりＮＡ及びＲ
ＮＡの特定のものを簡便かつ正確に測定しうる方法を提
供するものである。Detailed Description of the Invention (Industrial Application Field) Deoxylytic nucleic acids (DNA) and I having a specific structure
Measurement of Jg nucleic acid (RNA) is important in the field of biochemistry; for example, by measuring DNA in human serum, it is possible to test for viral infection or discover genetic diseases. The present invention is as follows: NA and R
The present invention provides a method for easily and accurately measuring a specific NA.
（従来の技術及び発明が解決しようとする問題点）
従来、このＤＮＡ又けＲＮＡの測定方法としては、試料
を変性処理して得た一本鎖ＤＮＡ （ Ｓ − ＤＮＡ
）又は一本鎖ＲＮＡ （ Ｓ − ＲＮＡ ）を一相
に結合させ、この固相にラノオアイソトーグを標識した
Ｓ−ＤＮＡ又はＳ − ＲＮＡを作用させて固相のＳ
− ＤＮＡ又はＳ − ＲＮＡとハイブリ，ドを形成さ
せてから未反応の標識Ｓ − ＤＮＡ又はＳ − ＲＮ
Ａを除去し、固相の放射線を測定する方法が行なわれて
いた。この方法は測定の際に固定化、洗浄等数多くの工
程を必要とし、特に試料の固定化に長時間を要するとこ
ろから操作の労力及び時間の両方に問題があった。(Problems to be Solved by the Prior Art and the Invention) Conventionally, as a method for measuring DNA and RNA, single-stranded DNA (S-DNA) obtained by denaturing a sample has been used.
) or single-stranded RNA (S-RNA) is bound to one phase, and S-DNA or S-RNA labeled with Lanoo isotologue is applied to this solid phase to bind S-RNA of the solid phase.
- unreacted labeled S-DNA or S-RN after hybridization with DNA or S-RNA;
A method has been used in which A is removed and the radiation of the solid phase is measured. This method requires many steps such as fixation and washing during measurement, and in particular, it takes a long time to fix the sample, which poses problems in terms of both operational labor and time.
一方、最近ＤＮＡ ｆ ｏ−ブを短かぐ切断してそのツ
端及びＪ端にエネルギートランスファーする螢光物質を
結合させ、ノいｆブリ、ドによりエネルギートランスフ
ァーを起こさせ、それによる螢光を測定してポリヌクレ
オチドを測定する技術が開発された（特開昭５８−４０
０９９号公報）。しかしながら、この方法はＤＮＡ ｆ
Ｄ−ブを短かく切断するために特異性が低下し、壕だ
、螢光量が少ないために感度が充分でない等の問題があ
った。On the other hand, recently, researchers have recently cut DNA fo-b into short pieces, bonded a fluorescent substance that transfers energy to the two ends and the J-end, caused energy transfer to occur, and measured the resulting fluorescence. A technique was developed to measure polynucleotides using
Publication No. 099). However, this method
There were problems such as a decrease in specificity due to the short cutting of the D-beam, and insufficient sensitivity due to the small amount of fluorescence.
本発明者らはこのような問題のない方法を開発すべく種
々検討の結果、予め標識された一本鎖ノリヌクレオチド
を固相に結合させておき、との固相に試料を変性処理さ
せて得た＝−杢調ポリヌクレオチドを作用させてハイブ
リ、ドを形成させ、ハイブリッドしたポリヌクレオチド
を制限酵素で切断する方法を案出し、その内存を歳に特
許出願（％願昭５８−１９９７０２号）した。しかしな
がら、この方法もまだ同相の分離が必要であり、特に大
量の検体を一度に処理する臨床分析等にあってはこの分
離操作が煩雑であった。The present inventors conducted various studies to develop a method free of such problems, and found that a pre-labeled single-stranded norinucleotide was bound to a solid phase, and the sample was denatured on the solid phase. He devised a method of reacting the obtained =- heathered polynucleotide to form a hybrid and cleaving the hybridized polynucleotide with a restriction enzyme, and filed a patent application (% Application No. 58-199702) for its existence. did. However, this method still requires in-phase separation, and this separation operation is particularly complicated in clinical analysis where a large amount of specimens are processed at once.
本発明はこのような問題点を解決するものであり、測定
対象の一本鎖ポリヌクレオチドとハイブリ、ドする一本
鎖ポリヌクレオチドに２種の発光物質を結合させ、その
間のエネルギー移動がこのポリヌクレオチドの切断によ
って変化することを利用している。The present invention solves these problems by bonding two types of luminescent substances to a single-stranded polynucleotide that hybridizes with the single-stranded polynucleotide to be measured, and the energy transfer between them is caused by this polynucleotide. It takes advantage of the change caused by cleavage of nucleotides.
（問題点を解決するだめの手段）
本発明は、測定対象である一本鎖ポリヌクレオチドを、
発光物質１又は発光物質１を直接又は間接的に発光させ
る物質とこの発光物質１■■■によって励起されて発光
する発光物質２とが結合され、かつ該測定対象二本鎖ポ
リヌクレオチドと二本鎖ポリヌクレオチドを形成しうる
一本鎖ポリヌクレオチドと接触させて二本鎖ポリヌクレ
オチドを形成させ、該二本鎖ポリヌクレオチドに二本鎖
ポリヌクレオチド制限酵素を作用させてこの二本鎖ポリ
ヌクレオチドを切断し、その後発光物質１又は発光物質
２の発光を測定することを特徴とするｒ１９リヌクレオ
チドの測定方法に関するものである。(Another means to solve the problem) The present invention provides a single-stranded polynucleotide to be measured,
A luminescent substance 1 or a substance that causes the luminescent substance 1 to emit light directly or indirectly and a luminescent substance 2 that emits light when excited by the luminescent substance 1 are combined, and the double-stranded polynucleotide to be measured and the double-stranded polynucleotide are bonded together. A double-stranded polynucleotide is formed by contacting a single-stranded polynucleotide capable of forming a stranded polynucleotide, and a double-stranded polynucleotide restriction enzyme is allowed to act on the double-stranded polynucleotide to convert the double-stranded polynucleotide into a double-stranded polynucleotide. The present invention relates to a method for measuring r19 polynucleotide, which comprises cleaving it and then measuring the luminescence of Luminescent Substance 1 or Luminescent Substance 2.
測定対象は一本鎖ポリヌクレオチド（以下、測定対象ポ
リヌクレオチドという。）である。測定対象ポリヌクレ
オチドにはＤＮＡ及びＲＮＡを含む゛。The measurement target is a single-stranded polynucleotide (hereinafter referred to as the measurement target polynucleotide). Polynucleotides to be measured include DNA and RNA.
試料中に含まれるポリヌクレオチドが二本鎖である場合
には水酸化ナトリウム溶液の添加などのアルカリ処理あ
るいは熱処理などにより一本鎖にしておく必要がある。If the polynucleotide contained in the sample is double-stranded, it is necessary to make it single-stranded by an alkali treatment such as addition of a sodium hydroxide solution or heat treatment.
試料の種類は問わないが、例えば人血清、尿１組織抽出
物などである。人血清などのようにポリヌクレオチドが
蛋白と結合しているおそれかある場合には試料をグロテ
アーゼ等で処理して蛋白を分離しておくのがよい。The type of sample does not matter; for example, it may be human serum or a urine tissue extract. When there is a possibility that polynucleotides may be bound to proteins, such as in human serum, it is preferable to separate the proteins by treating the sample with grotease or the like.
この測定対象ポリヌクレオチドと接触させる一本鎖ポリ
ヌクレオチド（以下、標識ポリヌクレオチドという。）
は発光物質ｌ又は発光物質１を直接又は間接的に発光さ
せる物質とこの発光物′Ｘ１■Ｉ■によって励起されて
発光する発光葡〜２とが結合されかつ該測定対象ポリヌ
クレオチドと二本鎖ポリヌクレオチドを形成しうるもの
である。A single-stranded polynucleotide (hereinafter referred to as a labeled polynucleotide) that is brought into contact with this polynucleotide to be measured.
is a double-stranded polynucleotide in which a luminescent substance 1 or a substance that causes the luminescent substance 1 to emit light directly or indirectly and a luminescent substance 2 that emits light when excited by the luminescent substance 1 It is capable of forming polynucleotides.
従って、この標識ポリヌクレオチドは測定対象ポリヌク
レオチドに対するグローブである。この標識ポリヌクレ
オチドは測定対象ポリヌクレオチドを含む二本鎖ポリヌ
クレオチドをアルカリ処理。Therefore, this labeled polynucleotide is a glove for the polynucleotide to be measured. This labeled polynucleotide is obtained by treating the double-stranded polynucleotide containing the polynucleotide to be measured with alkali.
熱処理などで変性させて得てもよく、あるいは測定対象
ポリヌクレオチド、 ＤＮａｓｅ Ｉ及びＤＮＡポリメ
ラーゼ１の存在下で各種ヌクレオチドを次々と結合させ
て得てもよい。It may be obtained by denaturing it by heat treatment or the like, or it may be obtained by sequentially bonding various nucleotides in the presence of the polynucleotide to be measured, DNase I, and DNA polymerase 1.
発光物質１は外部からの光を吸収して発光する螢光物質
又はリン光物質であってもよく、酸化反応等の化学反応
によって発光する化学発光物質あるいは生物発光物質で
おってもよい。The luminescent substance 1 may be a fluorescent substance or a phosphorescent substance that emits light by absorbing external light, or may be a chemiluminescent substance or a bioluminescent substance that emits light by a chemical reaction such as an oxidation reaction.
発光物質２は発光物質１■■ｌによって励起されて発光
するものであり、従って螢光物質又はリン光物質である
。The luminescent substance 2 is excited by the luminescent substance 1 and emits light, and is therefore a fluorescent substance or a phosphorescent substance.
発光物質１又は発光物質２に用いることができる螢光物
質は通常３２０ ｎｍ〜６００ｎｍ程度の波長で励起さ
れて螢光を発するものである。このような螢光物質の例
として、フルオレ、セイノ、ローダミン、グ／ジルクロ
ライド、フルオレスクアミン、クマリノ、アクリノン、
ＮＡＤＰＨ、ＮＡＤＨ、ベンゾオキサノアゾール、ト
リアリールメタン、ピレン類、これらの誘導体あるいは
活性化型のものなどを挙げることができる。The fluorescent substance that can be used as the luminescent substance 1 or the luminescent substance 2 is usually one that emits fluorescence when excited at a wavelength of about 320 nm to 600 nm. Examples of such fluorophores include fluorescein, seino, rhodamine, g/zyl chloride, fluorescuamine, coumarin, acrinon,
Examples include NADPH, NADH, benzoxanoazole, triarylmethane, pyrenes, and derivatives or activated forms thereof.
発光物質１に用いることができる化学発光物質の例とし
ては、ルミノール、イノルミノール、アクリノニウム、
ヒドロにルオキノド、ポルフィリン、イ／トレ／−３−
イルヒドロ″′Ｓ！ルオキシド、２．４．５１リフエニ
ルイミダゾール及びこれらの誘導体を挙げることができ
る。生物発光物質の例としてはルンフェラーゼによって
発光するルノフェリンを挙げることができる。Examples of chemiluminescent substances that can be used as the luminescent substance 1 include luminol, inluminol, acrinonium,
hydro, luoquinod, porphyrin, i/tre/-3-
Mention may be made of ylhydro''S! oxide, 2.4.51 rifhenylimidazole and derivatives thereof.An example of a bioluminescent substance is lunoferrin, which emits light by lumferase.
発光物質１に化学発光物質又は生物発光物質を用いる場
合には、標識物質にはこれらの発光物質を直接又は間接
的に発光させる物質を結合させることもできる。例えば
、発光物質■にルンフェリンを用いる場合にはルンフェ
ラーゼ又はその基質全結合させてこれらの作用によりル
ン７エリ／を発光させることができる。When a chemiluminescent substance or a bioluminescent substance is used as the luminescent substance 1, a substance that causes these luminescent substances to emit light can be bound to the labeling substance directly or indirectly. For example, when lumferin is used as the luminescent substance (1), lumferin can be fully bound to lumferase or its substrate, and lumferin can be caused to emit light by these actions.
本発明の方法においては、発光物質】■■−によって発
光物質２が励起されるのであるから両者の組合せはこの
ような関係が成立するものが選択されなければならない
。このような組合せの例としてフルオレ、セイ／とロー
ダミンを挙げることができる。フルオレ、ナインは４３
０〜５３０ ｎｍの波長の光により励起され５００〜５
６０ ｎｍの波長の螢光を発する。一方、ローダミンは
５３０〜５８０ ｎｍの波長の光により励起され５６０
〜６２０ｎ＋’ｎの波長の螢光を発する。従って、この
両者の混合物に４３０〜５３０ ｎｍの光を照射すると
５００〜５６０ｎｍの螢光に加えて５６０〜６２００ｍ
の螢光も現われる。In the method of the present invention, since the luminescent substance 2 is excited by the luminescent substance [■■-], the combination of the two must be selected such that such a relationship is established. Examples of such combinations include fluorescein, fluorine, and rhodamine. Fluore, nine is 43
Excited by light with a wavelength of 0 to 530 nm, 500 to 5
It emits fluorescent light with a wavelength of 60 nm. On the other hand, rhodamine is excited by light with a wavelength of 530 to 580 nm and produces 560
It emits fluorescent light with a wavelength of ~620n+'n. Therefore, when a mixture of the two is irradiated with light of 430 to 530 nm, in addition to the fluorescent light of 500 to 560 nm, it emits light of 560 to 6200 nm.
Fluorescence also appears.
発光物質１又はこれを直接又は間接的に発光婆せる物質
と発光物ｗ２とを標識ポリヌクレオチドとなる一重鎖ポ
リヌクレオチドに結合させる方法は公知の方法によれば
よく、例えば、モノヌクレオチドに発光物質を導入して
修飾型ヌクレオチド。The method of binding the luminescent substance 1 or a substance that can directly or indirectly increase luminescence and the luminescent substance w2 to a single-stranded polynucleotide serving as a labeled polynucleotide may be performed by a known method. modified nucleotides by introducing them.
とし、これに未修飾のモノヌクレオチドを混合してニッ
クトラ／スレー／カン法あるいはポリヌクレオチドの化
学的合成法等によってポリヌクレオチドにすればよい。This may be mixed with an unmodified mononucleotide to form a polynucleotide by the Nicktra/Three/Kan method or a chemical polynucleotide synthesis method.
そのほか、二、クトラ／スレーンヨン法や太陽菌を用い
るＣＤＮＡ作製法等によりポリヌクレオチドを作製し、
一方発光物質を活性化しておいてその活性基をポリヌク
レオチドと反応させてもよい。発光物質の結合方法とし
ては、ポリヌクレオチドに直接結合させる方法のほか、
ビオチ／又はアビツノを一方をポリヌクレオチドに結合
させもう一方を発光物質に結合させてこのビオチン−ア
ビノンの反応を利用して結合させるような間接的な方式
をとってもよい。In addition, polynucleotides are produced by the Kutra/Slaneyon method or the CDNA production method using solar fungi.
On the other hand, the luminescent substance may be activated and its active group reacted with the polynucleotide. Methods for binding luminescent substances include direct binding to polynucleotides,
An indirect method may be used in which one side of biotin/avinon is bonded to a polynucleotide and the other side is bonded to a luminescent substance, and the bond is made using this biotin-avinon reaction.
本発明の方法においては、発光物質１がら発光物質２へ
エネルギートランスファーを起こさせるところに特徴が
あり、このエネルギートランスファーを効率よく起こさ
せるためにポリヌクレオチドにおける発光物質１と発光
物質２との間隔をｓ ＯＸ以下、従って２４〜２６塩基
以内になるように両者を結合させることが好ましい。The method of the present invention is characterized in that energy transfer occurs from luminescent substance 1 to luminescent substance 2, and in order to cause this energy transfer to occur efficiently, the distance between luminescent substance 1 and luminescent substance 2 in the polynucleotide is adjusted. It is preferable that the two be bonded in such a manner that the number of bases is equal to or less than s OX, therefore, within 24 to 26 bases.
このような標識ポリヌクレオチドを測定対象ポリヌクレ
オチドと接触させて二本鎖ポリヌクレオチドを形成させ
る。接触時間は通常は測定対象ポリヌクレオチドが［２
ポリヌクレオチドと充分に反応して・・イブリッドを形
成しうる程度であるが、例えば０５〜４０時間程度が適
当である。温度は２０〜７０℃程度、ＰＨは５〜９程度
がよい。Such a labeled polynucleotide is brought into contact with a polynucleotide to be measured to form a double-stranded polynucleotide. The contact time is usually determined when the polynucleotide to be measured is [2
The appropriate time is, for example, about 0.5 to 40 hours, which is enough to sufficiently react with the polynucleotide to form a hybrid. The temperature is preferably about 20 to 70°C, and the pH is about 5 to 9.
二本鎖＋？ポリヌクレオチド形成させたのちはこれに二
本鎖、ｔ？ ｌ）ヌクレオチド制限酵素（以下、単に制
限酵素という。）を作用させる。この制限酵素ハ二木調
ポリヌクレオチドにのみ特異的に作用するものがよく、
まだ、認識ポリヌクレオチド鎖のあまり長くないものの
ほうが好ましい。制限酵素は１種のみでなく、２種以上
を併用してもよい。Double strand +? After forming a polynucleotide, this is double-stranded, t? l) Allow nucleotide restriction enzymes (hereinafter simply referred to as restriction enzymes) to act. It is best to use a restriction enzyme that specifically acts only on Haniki-style polynucleotides.
Still, less long recognition polynucleotide chains are preferred. Not only one restriction enzyme but also two or more restriction enzymes may be used in combination.
制限酵素を作用させる時期は通常は測定対象ポリヌクレ
オチドと標識ポリヌクレオチドとの反応終了後であるが
、」す定対象ポリヌクレオチドと同時あるいはその前に
反応系に添加しておいてもよい場合もちる。Restriction enzymes are normally allowed to act after the reaction between the polynucleotide to be measured and the labeled polynucleotide has completed, but in some cases it may be added to the reaction system at the same time as or before the polynucleotide to be measured. Ru.
制限啼素を作用させたのちは発光物質】又は発光物質２
０発光を測定する。測定は通常の螢光光度計を用いてそ
の強度を測定すればよい。Luminescent substance] or Luminescent substance 2 after acting with limiting nitrogen
Measure 0 emission. The intensity may be measured using an ordinary fluorophotometer.
（作用）
標Ｒポリヌクレオチドは測定対象ポリヌクレオチドとハ
イブリッドを形成する。ハイブリッドを形成することに
よって制限酵素が働いてこの二本鎖ポリヌクレオチドを
切断する。その結果、標識ポリヌクレオチドに結合され
ている発光物質１と発光物質２が分断されて別個に溶液
中を動き壕わるようになり、エネルギートランスファー
が起こらなくなる。そこで、発光物質１の発光強度の減
少はなくなり、一方、゛発光物質２からの発光はなくな
る。(Function) The target R polynucleotide forms a hybrid with the polynucleotide to be measured. Upon hybridization, restriction enzymes work to cleave this double-stranded polynucleotide. As a result, luminescent substance 1 and luminescent substance 2 bound to the labeled polynucleotide are separated and move separately in the solution, so that no energy transfer occurs. Therefore, the luminescent intensity of the luminescent material 1 no longer decreases, and on the other hand, the luminescent material 2 no longer emits light.
本発明の方法においては発光物質１から発光物質２への
エネルギートランスファーを利用しており、発光物質ｌ
の発光波長と発光物質２の発光波長が異なるところから
両者の発光を別々に定量できる。その結果、先願の方法
と異なり固相と液相の分離及びそれに付随する洗浄操作
が不要になる。The method of the present invention utilizes energy transfer from the luminescent substance 1 to the luminescent substance 2, and the luminescent substance l
Since the emission wavelength of the luminescent substance 2 and the emission wavelength of the luminescent substance 2 are different, the luminescence of the two can be separately quantified. As a result, unlike the method of the prior application, separation of the solid phase and liquid phase and the accompanying washing operation are not necessary.
本発明の方法は従来の方法と異なり、測定対象ポリヌク
レオチドを固相に固定するという煩雑な操作がなく、先
願の方法に比しても固液分離操作が不要な点でさらに簡
便にされている。本発明の方法に用いる試薬、器具類は
キット化が容易であり、とのキットを使用することによ
ってＤＮＡ等のポリヌクレオチド°を実用的かつ簡便に
測定することができる。Unlike conventional methods, the method of the present invention does not require the complicated operation of immobilizing the polynucleotide to be measured on a solid phase, and is even simpler than the method of the previous application in that it does not require a solid-liquid separation operation. ing. The reagents and instruments used in the method of the present invention can be easily made into a kit, and by using the kit, polynucleotides such as DNA can be measured practically and easily.
（１） ＨＢＶ −ＤＮＡ ｆロープの調製５００ｍ１
！の慢性Ｂ型肝炎患者のプール血清を９００Ｏｒｐｍで
１５分間遠心し、得られた上清を４℃１００ＯＯＯＸ
＆で５時間超遠心しテＨＢＶ粒子を４し、トとして集め
た。この被レットをＯ，］ＭＮａＣ１、１ｍＭ ＥＤＴ
Ａ 、 ０．１ ％２−メルカグトエタノール及び０１
チＢＳＡを含む０．０，１Ｍトリス−塩酸緩衝液（ｐＨ
７，５） ］、 Ｏｔｔｒｌに溶かし、このウィルス溶
液のうち５ ｍｌ ｆ保存し、残５ ｒｐｒｌを］ ０
００００ Ｘ ｇで再度５時間超遠心して被レットを得
だ。(1) Preparation of HBV-DNA f-rope 500ml
! Pooled serum from patients with chronic hepatitis B was centrifuged at 900 rpm for 15 minutes, and the resulting supernatant was heated at 100 OOOX at 4°C.
The HBV particles were ultracentrifuged for 5 hours at 400° C. and collected as 400° C. O, ]MNaC1, 1mM EDT
A, 0.1% 2-mercagutoethanol and 01
0.0,1M Tris-HCl buffer (pH
7,5) ], dissolve in Ottrl, save 5 ml of this virus solution, and save the remaining 5 rprl] 0
Ultracentrifugation was performed again at 0,000 x g for 5 hours to obtain pellets.
この被レットを０５％ＮＰ−４０を含むｌ ＯｍＭ ）
リス−塩酸、０．１ Ｍ ＮａＣ４ｐ８７．５溶液２０
０ μｔで処理し、ＤＮＡｙｆｆｌＪメラーゼを活性化
した。この溶液Ｋ １ ｍＭ ｄＡＴＰ 、 ］、
ｍＭ ｄＴＴＰ 、 ２．５ ａＭ ３２ＰｄＧＴＰ
、２、５ ａＭ ＰｄＣＴＰを含む０．０８ Ｍ Ｍ
ｇＣｌ２０．２ Ｍ ）リス緩衝液（ＰＨ７，５） ｓ
ｏμｔを加、えて３時間加温した。この溶液を３０チ
ンニークロース溶液の入った遠心チューブに重層し、５
Ｗ６５０−ター（べ。This coating was mixed with lOmM containing 05% NP-40).
Lith-hydrochloric acid, 0.1 M NaC4p87.5 solution 20
0 μt to activate DNAyfflJ merase. This solution K 1 mM dATP, ],
mM dTTP, 2.5 aM 32PdGTP
, 0.08 M M containing 2,5 aM PdCTP
gCl20.2M) Liss buffer (PH7,5)s
oμt was added and heated for 3 hours. This solution was layered in a centrifuge tube containing 30% Chinny Claus solution, and
W650-tar (beta).
クマ７社製）を用い５００００ｒｐｍで３時間遠心して
４し、トを得た。このぜし、トをグロナーゼで処理し、
得られた溶液をフェノールで２回抽出処理した。抽出液
を５〜２０％／ユークロースグラノエントで５００００
ｒｐｍＫて３時間遠心して１５３ ＰＤＮＡ分画を集
めこれをプールした。この分画から］ ５ Ｓ ”ＰＤ
ＮＡをエタノールを用いて沈澱させ、乾燥して目的のＨ
ＢＶ −ＤＮＡを得た。The mixture was centrifuged at 50,000 rpm for 3 hours using a microcomputer (manufactured by Kuma 7) to obtain a sample. After this, the tuna was treated with glonase,
The resulting solution was extracted twice with phenol. Extract 5-20%/Eucrose granoent 50,000
The mixture was centrifuged at rpmK for 3 hours, and 153 PDNA fractions were collected and pooled. From this fraction] 5S”PD
NA is precipitated using ethanol and dried to obtain the desired H
BV-DNA was obtained.
（２） 二、クトランスレー／ヨン法による修飾型ＨＢ
Ｖ −ＤＮＡの調製
次に、５ ｍＭＭｇｃｔ２．１０ ｍＭ２−１ルカ７”
ト：ｒ−タ／−ル、５ μＭｄＴＴＰ、 ５ μＭｄ
ＧＴＰ１５ ｔｔＭｄｃＴＰ。(2) 2. Modified HB by cutranthreeth/yon method
Preparation of V-DNA Next, 5 mM Mgct2.10 mM2-1 Luca7”
TTP: r-tal/-tal, 5 μMdTTP, 5 μMd
GTP15 ttMdcTP.
５ μＭｄＡＴＰ、 １０１１Ｍ７ミノヘキシルｄＡ
ＴＰ及び】０ＭＭアミノヘキソルｄＣＴＰ （５含む５
０ｍＭトリス−ＨＣ４緩衝液（ｐＨ７，５） ｌ ＯＯ
μｔにャｄ ５ ＨＢＶ−ＤＮＡ１ μＬ ＤＮａｓｅ
Ｉ １００ ｐｉｔ及びＤＮＡポリメラーゼ１１００
ｐｇを加えて１５℃で９０分間インキュベートした。5 μM dATP, 1011M7 minohexyl dA
TP and ]0MM aminohexol dCTP (contains 5
0mM Tris-HC4 buffer (pH 7,5) l OO
μtNyad 5 HBV-DNA1 μL DNase
I 100 pit and DNA polymerase 1100
pg was added and incubated at 15°C for 90 minutes.
この溶液をやはりフェノールで抽出し、５ｅｐｈａｄｅ
ｘ Ｇ −５０カラムで精製して修飾型ＨＢＶ −ＤＮ
Ａを得た。This solution was also extracted with phenol and
x G-50 column to purify modified HBV-DN
I got an A.
この二本鎖ＨＢＶ−ＤＮＡ １ ｍ９（’ １ ｍｌ
）とＨＢＶ−ＤＮＡを導した一木調Ｍ１３−ファノＤＮ
Ａ ５ ｍｔｉ （２” ） Ｋホルムアミド１３ ａ
ｔ ｆ加え、５分間この混合液を沸とうさせた。次に、
これに２ ｒｔｒｌの緩衝液（０，０７モル／ｌ、トリ
スーＨＣ４２モル／ ｌ ＮａＣ２，ｌ ５ｍＭ
ＥＤＴＡ ｐＨ７，５） ’に加え、５０℃で４時間そ
してさらに６０℃で１時間加温した。This double-stranded HBV-DNA 1 m9 (' 1 ml
) and Ichikicho M13-Fano DN that led to HBV-DNA
A 5 mti (2”) K formamide 13 a
tf was added and the mixture was boiled for 5 minutes. next,
To this was added 2 rtrl of buffer (0,07 mol/l, Tris-HC42 mol/l NaC2,l 5mM
EDTA pH 7,5)' and heated at 50°C for 4 hours and further at 60°C for 1 hour.
この反応液ｆ Ｂｌｏ−Ｇｅ１ Ａ ５０ ｍでダルｐ
過し、ハイブリ、ドしたＤＮＡと未反応のＤＮＡ ｉ分
離した。This reaction solution f Blo-Ge1 A was heated at 50 m
The hybridized, hybridized DNA and unreacted DNA were separated.
ボイド分画の近くに溶出される最初のピークを分画し、
これにＮａＣ４がＯ，１Ｍになるように粉末を加えて溶
かした。そして、さらに１００％エタノールを溶液１ｍ
εに対し２倍の比率（２＋＋ｌ）で加え、−７０℃で２
時間放置した。次に、］、７０００Ｘ、９で１０分間遠
心し、エタノール沈殿物ｆ ５ Ｑ ｍｌの０． ｌ
Ｎ ＮａＯＨ、０，２５ｍＭ ＥＤＴＡ、、 ０．００
１ ％フェノールレッドで溶解した。ただちにこれをＢ
ｌｏ−Ｃｅ１Ａ５０でグル濾過し、目的の一木調ＨＢＶ
−ＤＮＡを得た。Fractionate the first peak that elutes near the void fraction,
Powder was added to this so that NaC4 was O.1M and dissolved. Then, add 1 m of 100% ethanol solution.
Add at twice the ratio (2++l) to ε, and add 2 at -70℃.
I left it for a while. Then, centrifuge for 10 minutes at 7,000X, 9], and add 0.0% of the ethanol precipitate f5Q ml. l
N NaOH, 0.25mM EDTA, 0.00
Dissolved with 1% phenol red. Immediately B
Glu filtration with lo-Ce1A50 to obtain the target Ichikicho HBV
- DNA was obtained.
（３） 発光物質の導入
ローダミンイソチオ／アネート及びフレオレッセインイ
ソチオ／アネート各１．０μ９Ｆｔ含むＤＭＦ溶液１０
０μｔにこのＤＮＡ １・００μｇを含む２ｍεの０．
１Ｍ炭酸緩衝液ｐＨ１０，０を混合し、４℃で１８時間
反応させた。これをセフアゾ、リスＧ−５０でゲル濾過
し、フルオレッセイン及びローダミンを導入シたＨＢＶ
−ＤＮＡ １００ μｇ’を得た。(3) Introduction of luminescent substance 10 DMF solutions containing 1.0 μ9 Ft each of rhodamine isothio/anate and fluorescein isothio/anate
0.0 μt of 2 mε containing 1.00 μg of this DNA.
1M carbonate buffer pH 10.0 was mixed and reacted at 4°C for 18 hours. This was gel-filtered with cefazo and Risu G-50, and HBV was introduced with fluorescein and rhodamine.
-100 μg' of DNA was obtained.
（４） ヒト血清ＨＢＶ−ＤＮＡＯ測足ＨＢウィルス
性肝炎患者血清１００μ／！、ＫＯ，５ＮＨａＯＨ１０
０μｔを加えて室温で１０分間攪拌した。(4) Human serum HBV-DNAO measurement HB viral hepatitis patient serum 100μ/! ,KO,5NHaOH10
0 μt was added and stirred at room temperature for 10 minutes.
次に、０．５ Ｎ ＨＣｌ １００μｔを加えさらに２
００μｙｄのグロテイナーゼに溶液２００ ／１ｔを加
えて７０℃で１時間反応させた。（３）項で調製した発
光物質結合ＨＢＶ−ＤＮＡ Ｉ Ｏｎｇを含む溶液１０
０μｔを加えて３７℃で一夜放置した。これにｄ −Ｄ
ＮＡ制限酵素溶液（Ｂｇｌ ＩＩ 、 Ａｖａ ｎ ＋
Ｈａｅ Ｉｆ ＋ Ｈａｅ ｌｉｔ ＋ Ｈａｐ ｎ
＋Ｔ（ｉｎｃ［各Ｉ ＵＡＩ、 １０ ｍＭ ｈリス
−ＨＣｌ、７ｍＭＭｇＣ１２，７０ｍＭＮａｃｔ、 ７
ｍＭ ２−メルカプトエタノール、ｐＨ７，５）を１
．０ ｍｌ加えて３７℃で１時間反応させた。反応物に
’Ｉ−７０ｎｍの光を照射し、６００ ｎｍの螢光強度
を測定した。Next, add 100 μt of 0.5 N HCl and add 2
A solution of 200/1 t was added to 00 μyd of groteinase and reacted at 70°C for 1 hour. Solution 10 containing the luminescent substance-bound HBV-DNA I Ong prepared in section (3)
0 μt was added and left at 37° C. overnight. To this d-D
NA restriction enzyme solution (Bgl II, Ava n +
Hae If + Hae lit + Hap n
+T (inc [each I UAI, 10mM hLis-HCl, 7mM MgC12, 70mM Nact, 7
mM 2-mercaptoethanol, pH 7,5) at 1
．． 0 ml was added and reacted at 37°C for 1 hour. The reaction product was irradiated with 'I-70 nm light, and the fluorescence intensity at 600 nm was measured.
図面は得られた結果を示すものであり、縦軸は相対螢光
強度をそして横軸は血清の希釈度を表わしている。The figure shows the results obtained, with the vertical axis representing the relative fluorescence intensity and the horizontal axis representing the dilution of the serum.
次に本発明法及び従来のラノオアイントーグを用いた方
法で測定した各種ヒト血清のＨＢＶ −ＤＮＡ量を示す
。Next, the amounts of HBV-DNA in various human sera measured by the method of the present invention and the conventional method using Lanoaintog are shown.
本発明法 従来法
Ａ ］ｏｏｐｙ ’１１０ｐ、？Ｂ
ＮＤ ＮＤＣ２１０１／
２００／／
Ｄ ８９０ ／／ ８６０ ｔｔＥ
６０／／’ ７０〃Method of the present invention Conventional method A] oopy '110p,? B
ND NDC2101/
200 // D 890 // 860 ttE
60//' 70〃
図面は本発明法で測定した発光物質２の相対螢光強度と
血清希釈率との関係の一例を示すものである。The drawing shows an example of the relationship between the relative fluorescence intensity of the luminescent substance 2 and the serum dilution rate measured by the method of the present invention.
Claims (2)
物質１又は発光物質１を直接又は間接的に発光させる物
質とこの発光物質１によって励起されて発光する発光物
質２とが結合されかつ該測定対象一本鎖ポリヌクレオチ
ドと二本鎖ポリヌクレオチドを形成しうる一本鎖ポリヌ
クレオチドと接触させて二本鎖ポリヌクレオチドを形成
させ、該二本鎖ポリヌクレオチドに二本鎖ポリヌクレオ
チド制限酸素を作用させてこの二本鎖ポリヌクレオチド
を切断し、その後発光物質１又は発光物質２の発光を測
定することを特徴とするポリヌクレオチドの測定方法(1) A single-stranded polynucleotide to be measured is bound to a luminescent substance 1 or a substance that directly or indirectly causes the luminescent substance 1 to emit light, and a luminescent substance 2 that emits light when excited by the luminescent substance 1, and The single-stranded polynucleotide to be measured is brought into contact with a single-stranded polynucleotide that can form a double-stranded polynucleotide to form a double-stranded polynucleotide, and the double-stranded polynucleotide is subjected to double-stranded polynucleotide limiting oxygen. A method for measuring a polynucleotide, which comprises: cleaving the double-stranded polynucleotide by causing the double-stranded polynucleotide to act, and then measuring the luminescence of the luminescent substance 1 or the luminescent substance 2.
項記載の測定方法(2) Claim 1 in which the luminescent substance 2 is a fluorescent substance
Measurement method described in section
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59123757A JPS613063A (en) | 1984-06-18 | 1984-06-18 | Measurement of polynucleotide utilizing luminous substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59123757A JPS613063A (en) | 1984-06-18 | 1984-06-18 | Measurement of polynucleotide utilizing luminous substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS613063A true JPS613063A (en) | 1986-01-09 |
| JPH0515439B2 JPH0515439B2 (en) | 1993-03-01 |
Family
ID=14868543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59123757A Granted JPS613063A (en) | 1984-06-18 | 1984-06-18 | Measurement of polynucleotide utilizing luminous substance |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS613063A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015270A1 (en) * | 1994-11-16 | 1996-05-23 | Perkin-Elmer Corporation | Self-quenching fluorescence probe and method |
| EP0878554A3 (en) * | 1997-05-13 | 2001-11-28 | Becton, Dickinson and Company | Detection of nucleic acids by fluorescence quenching |
| WO2003035864A1 (en) * | 2001-10-26 | 2003-05-01 | Matsushita Electric Industrial Co., Ltd. | Method of detecting target nucleic acid and nucleic acid probe |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6379888B1 (en) | 1999-09-27 | 2002-04-30 | Becton, Dickinson And Company | Universal probes and methods for detection of nucleic acids |
-
1984
- 1984-06-18 JP JP59123757A patent/JPS613063A/en active Granted
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015270A1 (en) * | 1994-11-16 | 1996-05-23 | Perkin-Elmer Corporation | Self-quenching fluorescence probe and method |
| EP0972848A3 (en) * | 1994-11-16 | 2000-10-04 | Perkin-Elmer Corporation | Self-quenching fluorescence probe and method |
| EP0878554A3 (en) * | 1997-05-13 | 2001-11-28 | Becton, Dickinson and Company | Detection of nucleic acids by fluorescence quenching |
| WO2003035864A1 (en) * | 2001-10-26 | 2003-05-01 | Matsushita Electric Industrial Co., Ltd. | Method of detecting target nucleic acid and nucleic acid probe |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0515439B2 (en) | 1993-03-01 |
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