JPH11318497A - Method for mixing upper and lower layer solutions using sinker for promoting hot start of polymerase reaction - Google Patents
Method for mixing upper and lower layer solutions using sinker for promoting hot start of polymerase reactionInfo
- Publication number
- JPH11318497A JPH11318497A JP14042898A JP14042898A JPH11318497A JP H11318497 A JPH11318497 A JP H11318497A JP 14042898 A JP14042898 A JP 14042898A JP 14042898 A JP14042898 A JP 14042898A JP H11318497 A JPH11318497 A JP H11318497A
- Authority
- JP
- Japan
- Prior art keywords
- lower layer
- solution
- reaction
- oils
- fat
- 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.)
- Withdrawn
Links
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は一種のポリメラーゼ
反応のホットスタートを促進する沈子を利用した上下層
溶液混合方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mixing upper and lower layer solutions using a sediment which promotes a kind of hot start of a polymerase reaction.
【０００２】[0002]
【従来の技術】油脂を中間として反応液体を上下層に分
けることを利用し、ポリメラーゼ反応（ｐｏｌｙｍｅｒ
ａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ）のホットスタ
ート（ｈｏｔ ｓｔａｒｔ）効果或いは逆転写（ｒｅｖ
ｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ）反応が達成さ
れている。その原理は、高融点油脂（通常融点４０−７
０℃）の固体から液体への形態の変化が丁度反応時の温
度変化と符合することを利用しており、即ち、反応管ｅ
（図１及び図２参照）内の中層の油脂ｂがポリメラーゼ
反応の前に固体であり、上下層溶液ａ、ｃを隔離してい
るが、温度が上昇開始してポリメラーゼ反応を進行する
時、固体の油脂ｂが変化し、且つ油脂ｂの密度が水より
小さく液化後に液面に浮くことから、上下層溶液ａ、ｃ
を自然に混合させてポリメラーゼ反応を進行できるよう
にするものである。2. Description of the Related Art A polymerase reaction (polymer) is carried out utilizing the separation of a reaction liquid into upper and lower layers with an oil or fat as an intermediate.
hot start effect of reverse chain reaction or reverse transcription (rev)
erase transcription reaction has been achieved. The principle is that high melting point fat (normally 40-40
(0 ° C.) utilizing the fact that the change in form from solid to liquid exactly matches the temperature change during the reaction, ie, the reaction tube e
(See FIGS. 1 and 2) The middle layer fat b is solid before the polymerase reaction and separates the upper and lower layer solutions a and c, but when the temperature starts to rise and the polymerase reaction proceeds, Since the solid fat b changes and the density of the fat b is lower than water and floats on the liquid surface after liquefaction, the upper and lower layer solutions a and c
Are naturally mixed to allow the polymerase reaction to proceed.
【０００３】ただし、上層の液体の重力、及び油脂と管
壁の付着力により液化した油脂がその密度が水より小さ
くとも液面に浮かばず、中層に滞留し、上下層液の混合
を妨げてポリメラーゼ反応が進行不能となることがよく
ある。However, even if the density of the oil or fat is smaller than water, the oil or fat liquefied by the gravity of the liquid in the upper layer and the adhesion between the oil and the pipe wall does not float on the liquid surface, stays in the middle layer and hinders the mixing of the upper and lower layer liquids. Often, the polymerase reaction cannot proceed.
【０００４】[0004]
【発明が解決しようとする課題】本発明は、一種のポリ
メラーゼ反応のホットスタートを促進する沈子を利用し
た上下層溶液混合方法を提供することを課題とし、該方
法では、沈子の下沈作用を利用し、反応管内の溶液の流
動をトリガして、上下層の溶液の混合を促進し、並びに
油脂を管底に沈めるか或いは油脂に液面にて火山状の開
口を形成させることで、油脂が反応完了後に管内の溶液
の吸い取りを妨害しないようにする。SUMMARY OF THE INVENTION An object of the present invention is to provide a method of mixing upper and lower layer solutions using a sediment which promotes a kind of hot start of a polymerase reaction. By utilizing the action, the flow of the solution in the reaction tube is triggered to promote the mixing of the upper and lower layers of the solution, and the oil or fat is submerged at the bottom of the tube or the oil or fat forms a volcanic opening at the liquid level. Ensure that the oils and fats do not interfere with the uptake of the solution in the tube after the reaction is complete.
【０００５】[0005]
【課題を解決するための手段】請求項１の発明は、上層
溶液と下層溶液とを中層の油脂層で隔離した状態で収容
する反応管中にあって、一種の、ポリメラーゼ反応のホ
ットスタートを促進する上下層溶液混合方法とされて、
以下の１から４のステップ、即ち、 １．沈子が上層溶液に投入されるステップ ２．温度が上昇させられ、中層の油脂Ｂが液化開始しポ
リメラーゼ反応が開始されるステップ ３．沈子が下層に沈み、それに伴い油脂が下降し、上層
溶液と下層溶液が混合開始するステップ ４．油脂が反応管の管底壁に付着し、凝集力により反応
管の管底に滞留するか、油脂が浮上して火山口状の開口
を液面に形成するステップ 以上のステップを包括することを特徴とする、ポリメラ
ーゼ反応のホットスタートを促進する沈子を利用した上
下層溶液混合方法としている。According to the present invention, there is provided a reaction tube for accommodating an upper solution and a lower solution in a state of being separated by an intermediate oil layer. Promoted upper and lower layer solution mixing method,
Steps 1 to 4 below: 1. The step of introducing the sludge into the upper solution. Step 2. The temperature is raised, the fat B in the middle layer starts to liquefy, and the polymerase reaction starts. 3. A step in which the sediment sinks to the lower layer, the fat and oil descends, and the upper layer solution and the lower layer solution start mixing. Steps in which oils and fats adhere to the tube bottom wall of the reaction tube and stay at the tube bottom of the reaction tube due to cohesive force, or steps in which the oils and fats float to form a volcano-mouth-shaped opening on the liquid surface. The method is characterized by a method of mixing upper and lower layer solutions using a pellet which promotes a hot start of a polymerase reaction.
【０００６】請求項２の発明は、前記沈子がシリカビー
ズ或いはシリカ粒とされることを特徴とする、請求項１
に記載のポリメラーゼ反応のホットスタートを促進する
沈子を利用した上下層溶液混合方法としている。According to a second aspect of the present invention, the sediment is silica beads or silica particles.
And a method of mixing upper and lower layer solutions using a pellet for promoting hot start of the polymerase reaction described in (1).
【０００７】[0007]
【発明の実施の形態】本発明はポリメラーゼ反応のホッ
トスタートを促進する沈子を利用した上下層溶液混合方
法を提供し、本発明では沈子の下沈作用を利用して油脂
層を反応管内での下向きに流動させて底層に留めるか或
いは油脂層に液面で火山口状の開口を形成させること
で、反応管内の溶液の吸い取りを妨害しないようにして
いる。DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for mixing upper and lower layer solutions using a sediment which promotes hot start of a polymerase reaction. In the present invention, an oil / fat layer is formed in a reaction tube by utilizing a sedimentation effect of a sediment. By flowing the solution downward in the bottom and keeping it in the bottom layer, or by forming a volcanic pit-like opening in the oil and fat layer at the liquid level, the absorption of the solution in the reaction tube is not hindered.
【０００８】[0008]
【実施例】実施例１：反応管Ｅ（図３参照）内にて油脂
Ｂを介在させることを利用して反応管Ｅ内の上層溶液Ａ
と下層溶液Ｃを隔離する。本発明では沈子Ｄを利用して
いるが、該沈子Ｄの反応管Ｅ内での下沈過程は以下の四
つのステップからなる。 １．沈子Ｄが上層溶液Ａに投入される（図３参照）。該
沈子Ｄはシリカビーズ（ｓｉｌｉｃａ ｂｅａｄ）或い
はシリカ粒とされる。 ２．温度が上昇させられ、中層の油脂Ｂが液化開始しポ
リメラーゼ反応が開始される（図４参照）。 ３．沈子Ｄが下層に沈み、それに伴い油脂Ｂが下降し
（図４参照）、上層溶液Ａと下層溶液Ｃが混合開始す
る。 ４．油脂Ｂが反応管Ｅの管底壁に付着し、凝集力により
反応管Ｅの管底に滞留し（図５参照）、別に油脂Ｂは密
度の関係から浮上して火山口状の開口Ｆを液面に形成し
うる（図６参照）。EXAMPLE 1 An upper layer solution A in a reaction tube E utilizing the interposition of fats and oils B in a reaction tube E (see FIG. 3).
And the lower layer solution C. In the present invention, the sediment D is used. The process of sedimentation of the sediment D in the reaction tube E includes the following four steps. 1. Pellet D is introduced into upper layer solution A (see FIG. 3). The sediment D is a silica bead or a silica particle. 2. The temperature is increased, and the fat B in the middle layer starts to liquefy and the polymerase reaction starts (see FIG. 4). 3. The sediment D sinks to the lower layer, and the fat B falls accordingly (see FIG. 4), and the upper solution A and the lower solution C start mixing. 4. The fat B adheres to the bottom of the reaction tube E and stays at the bottom of the reaction tube E due to cohesion (see FIG. 5). It can be formed on the liquid surface (see FIG. 6).
【０００９】実施例２： １．３０ｕｌの溶液Ｃが０．２ｍｌのポリメラーゼ反応
専用の反応管Ｅに投入される。 ２．２０ｕｌの液状の油脂Ｂ、例えばパラフィン類、或
いはワックスなど融点５０−６０℃、密度約０．９の油
脂が加えられる。 ３．油脂の凝固を待って、３０ｕｌの上層溶液Ａが加え
られる。 ４．沈子Ｄとして、例えば密度約２．５、直径１．５−
２．０ｍｍのシリカビーズ、或いはその他の粒子が投入
される。 ５．反応管Ｅが適当な加熱器、例えばサーモサイクラー
（ｔｈｅｒｍｏｃｙｃｌｅｒ）中に置かれる。 ６．プログラム設定される（例えば逆転写／ポリメラー
ゼ反応或いはＨｓポリメラーゼ反応）。 ７．プログラムが起動される。Example 2: 1.30 ul of solution C is charged into 0.2 ml of a reaction tube E dedicated to a polymerase reaction. 2.20 ul of liquid fat B, for example paraffins or wax, having a melting point of 50-60 ° C. and a density of about 0.9 is added. 3. Waiting for the fat to coagulate, 30 ul of top solution A is added. 4. As the seeds D, for example, a density of about 2.5 and a diameter of 1.5-
2.0 mm silica beads or other particles are charged. 5. Reaction tube E is placed in a suitable heater, for example, a thermocycler. 6. Programmed (eg, reverse transcription / polymerase reaction or Hs polymerase reaction). 7. The program starts.
【００１０】室温にあるか或いは逆転写反応が進行され
る時には油脂は固態相を保持し、上層溶液と下層溶液が
隔離され、ポリメラーゼ反応が開始されて温度が上昇し
た時（通常９４℃）、固体の油脂が液化し、沈子の重力
により油脂に下に凹む現象が発生し、並びに油脂が管壁
に沿って下向きに移動し（反応管壁は例えばポリプロピ
レンで製造され親油性或いは疎水性のものとされ、油脂
相が良好な親和力を保持しうるため）、沈子が継続して
下沈すると以下の二種類の状況が発生しうる。 １．沈子が油脂相を突き抜け、油脂相の凝集力が消失
し、油脂が管壁に付着すし、並びに密度の関係から液面
に浮上し、火山口状の開口を形成する。 ２．沈子が油脂相を突き抜けるか或いは突き抜けず、油
脂層の下凹部分が管底壁に接触し並びに管底壁に付着
し、また油脂自体の凝集力により、油脂が管底に凝集す
る。At room temperature or when the reverse transcription reaction proceeds, the fat or oil retains a solid phase, the upper layer solution and the lower layer solution are isolated, and when the polymerase reaction is started and the temperature rises (usually 94 ° C.), Solid fats and oils are liquefied and the gravitational force of the sediment causes a phenomenon that the oils and fats dent downward, and the fats and oils move downward along the tube wall (the reaction tube wall is made of, for example, polypropylene and is made of If the oil phase can keep good affinity), the following two situations can occur when the sediment continues to sink down. 1. The sediment penetrates the oil phase, the cohesive force of the oil phase disappears, the oil adheres to the tube wall, and floats on the liquid surface due to the density, forming a volcanic pit-like opening. 2. The sediment penetrates or does not penetrate the oil phase, the lower concave portion of the oil layer comes into contact with the tube bottom wall and adheres to the tube bottom wall, and the oil and fat coagulate on the tube bottom by the cohesive force of the oil and fat itself.
【００１１】[0011]
【発明の効果】本発明によると、沈子と油脂層の下沈過
程で、上層溶液と下層溶液も同時に混合され、ポリメラ
ーゼ反応が順調に進行され、沈子の利用により油脂層反
応管が広い利用価値を有するものとなり、油脂層が上層
溶液と下層溶液の隔離専用に使用されて沈子により上下
層の溶液の混合が促進され、並びに油脂層が反応完了後
に反応管内の溶液の吸い出しを妨害しない。According to the present invention, the upper layer solution and the lower layer solution are simultaneously mixed during the sedimentation of the sediment and the oil layer, the polymerase reaction proceeds smoothly, and the oil / fat layer reaction tube is widened by utilizing the sediment. The oil and fat layer is used exclusively for separating the upper layer solution and the lower layer solution, and the sediment promotes the mixing of the upper and lower layer solutions, and the oil and fat layer hinders the suction of the solution in the reaction tube after the reaction is completed. do not do.
【図１】従来の方法による、反応管内で固形油脂が上層
と下層溶液との間を隔てている状態表示図である。FIG. 1 is a view showing a state in which a solid fat separates an upper layer solution from a lower layer solution in a reaction tube according to a conventional method.
【図２】従来の方法による、反応管内で固形油脂が液化
し上層と下層溶液とが混合される状態表示図である。FIG. 2 is a view showing a state in which solid fats and oils are liquefied in a reaction tube and an upper layer solution and a lower layer solution are mixed according to a conventional method.
【図３】本発明の方法による、沈子が上層溶液にある状
態表示図である。FIG. 3 is a schematic view showing a state in which a seed is present in an upper solution according to the method of the present invention.
【図４】本発明の方法による、油脂が液化開始し沈子が
下沈する状態表示図である。FIG. 4 is a diagram showing a state in which fats and oils start to liquefy and sinks sink down according to the method of the present invention.
【図５】本発明の方法による、沈子が下沈し、上層と下
層の溶液が混合され、油脂層が管体にある状態表示図で
ある。FIG. 5 is a view showing a state in which the seeds are settled down, the upper layer solution and the lower layer solution are mixed, and the oil layer is in the tube according to the method of the present invention.
【図６】本発明の方法による、沈子が下沈し、上層と下
層の溶液が混合され、油脂層が液面にある状態表示図で
ある。FIG. 6 is a view showing a state in which the seeds are settled down, the upper layer solution and the lower layer solution are mixed, and the oil and fat layer is at the liquid level according to the method of the present invention.
Ａ 上層溶液 Ｂ 油脂 Ｃ 下層溶液 Ｄ 沈子 Ｅ 反応管 Ｆ 開口 ａ 上層溶液 ｂ 油脂 ｃ 下層溶液 ｅ 反応管 A upper layer solution B oil and fat C lower layer solution D sediment E reaction tube F opening a upper layer solution b oil and fat c lower layer solution e reaction tube
Claims (2)
隔離した状態で収容する反応管中にあって、一種の、ポ
リメラーゼ反応のホットスタートを促進する上下層溶液
混合方法とされて、以下の１から４のステップ、即ち、 １．沈子が上層溶液に投入されるステップ ２．温度が上昇させられ、中層の油脂Ｂが液化開始しポ
リメラーゼ反応が開始されるステップ ３．沈子が下層に沈み、それに伴い油脂が下降し、上層
溶液と下層溶液が混合開始するステップ ４．油脂が反応管の管底壁に付着し、凝集力により反応
管の管底に滞留するか、油脂が浮上して火山口状の開口
を液面に形成するステップ 以上のステップを包括することを特徴とする、ポリメラ
ーゼ反応のホットスタートを促進する沈子を利用した上
下層溶液混合方法。1. A method of mixing upper and lower layer solutions which promotes hot start of a polymerase reaction in a reaction tube containing an upper layer solution and a lower layer solution in a state separated by an intermediate oil layer, Steps 1 to 4 below: 1. The step of introducing the sludge into the upper solution. Step 2. The temperature is raised, the fat B in the middle layer starts to liquefy, and the polymerase reaction starts. 3. A step in which the sediment sinks to the lower layer, the fat and oil descends, and the upper layer solution and the lower layer solution start mixing. Steps in which oils and fats adhere to the tube bottom wall of the reaction tube and stay at the tube bottom of the reaction tube due to cohesive force, or steps in which the oils and fats float to form a volcano-mouth-shaped opening on the liquid surface. A method for mixing upper and lower layer solutions using a pellet that promotes hot start of a polymerase reaction.
とされることを特徴とする、請求項１に記載のポリメラ
ーゼ反応のホットスタートを促進する沈子を利用した上
下層溶液混合方法。2. The method of claim 1, wherein the precipitate is silica beads or silica particles. 2. The method of claim 1, wherein the precipitate is formed of silica beads or silica particles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14042898A JPH11318497A (en) | 1998-05-08 | 1998-05-08 | Method for mixing upper and lower layer solutions using sinker for promoting hot start of polymerase reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14042898A JPH11318497A (en) | 1998-05-08 | 1998-05-08 | Method for mixing upper and lower layer solutions using sinker for promoting hot start of polymerase reaction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11318497A true JPH11318497A (en) | 1999-11-24 |
Family
ID=15268466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14042898A Withdrawn JPH11318497A (en) | 1998-05-08 | 1998-05-08 | Method for mixing upper and lower layer solutions using sinker for promoting hot start of polymerase reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11318497A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8026068B2 (en) | 2002-01-08 | 2011-09-27 | Roche Molecular Systems, Inc. | Use of silica material in an amplification reaction |
-
1998
- 1998-05-08 JP JP14042898A patent/JPH11318497A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8026068B2 (en) | 2002-01-08 | 2011-09-27 | Roche Molecular Systems, Inc. | Use of silica material in an amplification reaction |
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