JPH0347097A - Hybridization method, gene mutation detection using same and apparatus therefor - Google Patents
Hybridization method, gene mutation detection using same and apparatus thereforInfo
- Publication number
- JPH0347097A JPH0347097A JP1178933A JP17893389A JPH0347097A JP H0347097 A JPH0347097 A JP H0347097A JP 1178933 A JP1178933 A JP 1178933A JP 17893389 A JP17893389 A JP 17893389A JP H0347097 A JPH0347097 A JP H0347097A
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- nucleic acid
- electrophoresis
- carrier
- acid probe
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- 102000039446 nucleic acids Human genes 0.000 claims abstract description 42
- 238000006243 chemical reaction Methods 0.000 claims abstract description 39
- 230000035772 mutation Effects 0.000 claims description 32
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- 239000000463 material Substances 0.000 claims description 2
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- 239000003298 DNA probe Substances 0.000 description 18
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- 238000002372 labelling Methods 0.000 description 4
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- 239000000020 Nitrocellulose Substances 0.000 description 3
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は核酸試料のハイブリダイゼーション方法、この
方法を用いた遺伝子変異検出方法及びその装置に関し、
特に高速で自動化容易な遺伝子変異検出方法及び装置に
関する。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for hybridizing a nucleic acid sample, a method for detecting genetic mutations using this method, and an apparatus for the same.
In particular, the present invention relates to a method and device for detecting genetic mutations that are fast and easy to automate.
核酸（ＤＮＡ又はＲＮＡ）試料又はＤＮＡ （ＲＮＡ）
プローブ（標的ＤＮＡ （ＲＮＡ）と相補的な塩基配列
を持つＤＮＡ （ＲＮＡ）断片）のいずれか一方を固相
に固定したハイブリダイゼーション反応を用いる従来の
遺伝子変異検出法は、プロシーデインゲス オブ ナチ
ュラルアカデミーオプ サイエンス ニー ニス ニー
、 ８０巻（１９８３年）第２７８頁から２８２頁（Ｐ
ｒｏｃ、Ｎａｔｌ、 Ａｃａｄ、Ｓｃｉ。Nucleic acid (DNA or RNA) sample or DNA (RNA)
Conventional genetic mutation detection methods that use a hybridization reaction in which one of the probes (a DNA (RNA) fragment with a complementary base sequence to the target DNA (RNA)) is immobilized on a solid phase are Op Science Nis Nis Ni, Vol. 80 (1983), pp. 278-282 (P.
roc, Natl, Acad, Sci.
ＵＳＡ、 ８０． （１９８３）、 ｐｐ、２７８〜２
８２）に記載されている。USA, 80. (1983), pp. 278-2.
82).
この方法は、まず、電気泳動によって分子量分離したＤ
ＮＡ断片試料をニトロセルロース膜上に転写、固定した
後、この膜をＤＮＡプローブを含む溶液に浸してハイブ
リダイゼーション反応を行なう。ハイブリダイゼーショ
ン反応では、塩基配列の相補性が高い程、ＤＮＡ断片試
料とＤＮＡプローブは強く結合し、高い温度でも解離す
ることがない、そこで、次に、ＤＮＡ断片試料がＤＮＡ
プローブと完全な相補性をもつ場合には解離せず、相補
性がないか又は相補性が不完全な場合には解離するよう
な温度で洗浄を行なう。ＤＮＡ断片試料がＤＮＡプロー
ブと完全な相補性を持つものである場合には、ＤＮＡプ
ローブは膜に結合したまま残って検出されるが、そうで
ない場合には、ＤＮＡプローブは膜から洗い流されて検
出されない。In this method, D is first separated by molecular weight by electrophoresis.
After transferring and fixing the NA fragment sample onto a nitrocellulose membrane, the membrane is immersed in a solution containing a DNA probe to perform a hybridization reaction. In a hybridization reaction, the higher the complementarity of the base sequences, the stronger the bond between the DNA fragment sample and the DNA probe, and they will not dissociate even at high temperatures.
Washing is carried out at a temperature that does not dissociate when the probe has complete complementarity with the probe, but dissociates when there is no complementarity or incomplete complementarity. If the DNA fragment sample has perfect complementarity with the DNA probe, the DNA probe remains bound to the membrane and can be detected; otherwise, the DNA probe is washed away from the membrane and cannot be detected. Not done.
以上のように、この方法では、ＤＮＡ断片試料がＤＮＡ
プローブと完全な相補性を持つか否かを判定できる。し
たがって、正常な標的遺伝子と完全な相補性を持つＤＮ
Ａ断片をＤＮＡプローブとすることにより、ＤＮＡ断片
試料中の標的遺伝子が正常なものか、あるいはポイント
ミューチージョン、挿入、欠失等の変異を含む異常なも
のかを判定でき、遺伝子の変異を検出できる。As described above, in this method, the DNA fragment sample is
It can be determined whether it has complete complementarity with the probe. Therefore, a DN with perfect complementarity with a normal target gene
By using Fragment A as a DNA probe, it is possible to determine whether the target gene in a DNA fragment sample is normal or abnormal, including mutations such as point mutations, insertions, and deletions. Can be detected.
〔発明が解決しようとする課題］
上記の従来法では、ハイブリダイゼーション反応がニト
ロセルロース膜（固相）に固定されたＤＮＡ断片試料と
、溶液中のＤＮＡプローブの受動的拡散によって起こる
ため、反応速度が遅いという問題点があった。また、反
応時及び洗浄時には、各々の溶液の注入、排出という自
動化しにくい動作が含まれているという問題点があった
。[Problems to be Solved by the Invention] In the above conventional method, the hybridization reaction occurs by passive diffusion of the DNA fragment sample immobilized on the nitrocellulose membrane (solid phase) and the DNA probe in the solution, so the reaction rate is low. The problem was that it was slow. Furthermore, there is a problem in that the reactions and cleaning involve operations that are difficult to automate, such as injecting and discharging each solution.
本発明の目的は、ハイブリダイゼーションの反応速度が
速く、しかも溶液の注入、排出等の自動化しにくい動作
の少ない、高速で自動化容易なハイブリダイゼーション
方法、該方法を用いた遺伝子変異検出法及びそれに用い
る装置を提供することにある。The purpose of the present invention is to provide a high-speed and easy-to-automate hybridization method that has a high hybridization reaction rate and requires few operations that are difficult to automate, such as injection and discharge of solutions, a method for detecting genetic mutations using the method, and a method for detecting genetic mutations using the method. The goal is to provide equipment.
上記目的を達成するために、本発明では、ＤＮＡプロー
ブを電気泳動担体に固定し、その上下に緩衝液を介して
２つの電極を配置して、電気泳動により核酸断片試料等
を強制的に移動させて、ハイブリダイゼーション反応や
洗浄を行なうようにした。In order to achieve the above object, in the present invention, a DNA probe is immobilized on an electrophoresis carrier, two electrodes are placed above and below the carrier via a buffer solution, and nucleic acid fragment samples, etc. are forcibly moved by electrophoresis. Then, hybridization reaction and washing were performed.
即ち、本発明は、核酸プローブと核酸試料のハイブリダ
イゼーション方法において、核酸プローブを電気泳動担
体中に固定し、核酸試料を電気泳動によって電気担体中
に移動せしめることを特徴とする核酸試料のハイブリダ
イゼーション方法である。このハイブリダイゼーション
方法によれば、ＤＮＡプローブを固定した電気泳動担体
上を核酸試料を強制的に移動させるものであるから、ハ
イブリダイゼーション反応が、上記従来法に比して速く
、この反応を短時間で完了することができる。That is, the present invention provides a method for hybridizing a nucleic acid probe and a nucleic acid sample, which is characterized in that the nucleic acid probe is immobilized in an electrophoretic carrier, and the nucleic acid sample is moved into the electrophoretic carrier by electrophoresis. It's a method. According to this hybridization method, the nucleic acid sample is forcibly moved on the electrophoresis carrier on which the DNA probe is immobilized, so the hybridization reaction is faster than in the conventional method, and the reaction can be carried out in a short time. It can be completed with.
さらに本発明は、核酸プローブと核酸試料のハイブリダ
イゼーション反応を用いた遺伝子変異検出法において、
核酸プローブを電気泳動担体中に固定し、核酸試料を電
気泳動によって電気泳動担体中に移動せしめてハイブリ
ダイゼーション反応を行わせ、上記核酸プローブと結合
しなかった上記核酸試料を電気泳動によって移動せしめ
上記電気泳動担体中から除去することを特徴とする遺伝
子変異検出方法である。Furthermore, the present invention provides a genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample.
A nucleic acid probe is immobilized in an electrophoretic carrier, a nucleic acid sample is moved into the electrophoretic carrier by electrophoresis to perform a hybridization reaction, and the nucleic acid sample that does not bind to the nucleic acid probe is moved by electrophoresis. This is a method for detecting genetic mutations characterized by removal from an electrophoresis carrier.
上記遺伝子変異検出法においては、２種類の核酸プロー
ブ、即ち、電気泳動担体に固定する核酸プローブ（固定
化プローブ）と、前記固定化プローブに結合した核酸試
料に更にハイブリダイズする標識化された第２の核酸プ
ローブ（標識プローブ）を用いて行うことができる。即
ち、この遺伝子変異検出法は、核酸プローブを電気泳動
担体中に固定し、核酸試料を電気泳動によって電気泳動
担体中に移動せしめてハイブリダイゼーション反応を行
わせ、上記核酸プローブと結合しなかった上記核酸試料
を電気泳動によって移動せしめて電気泳動担体中から除
去し、さらに標識核酸プローブを電気泳動によって電気
泳動担体中に移動せしめてハイブリダイゼーション反応
を行わせ、上記核酸プローブと結合しなかった上記標識
核酸プローブを電気泳動によって移動せしめて電気泳動
担体中から除去した後、上記核酸試料と結合した標識核
酸プローブの標識を検出することにより行うことができ
る。In the above gene mutation detection method, two types of nucleic acid probes are used: a nucleic acid probe (immobilized probe) that is immobilized on an electrophoresis carrier, and a labeled probe that further hybridizes to the nucleic acid sample bound to the immobilized probe. This can be carried out using the following nucleic acid probe (labeled probe). That is, in this gene mutation detection method, a nucleic acid probe is immobilized in an electrophoretic carrier, a nucleic acid sample is moved into the electrophoretic carrier by electrophoresis, a hybridization reaction is performed, and the above-mentioned nucleic acid probe that does not bind to the nucleic acid probe is transferred to the electrophoretic carrier. The nucleic acid sample is moved by electrophoresis and removed from the electrophoresis carrier, and the labeled nucleic acid probe is further moved into the electrophoresis carrier by electrophoresis to perform a hybridization reaction, and the label that does not bind to the nucleic acid probe is removed. This can be carried out by moving the nucleic acid probe by electrophoresis and removing it from the electrophoresis carrier, and then detecting the label of the labeled nucleic acid probe bound to the nucleic acid sample.
また、上記いずれの方法においてもハイブリダイゼーシ
ョン反応を行わせた後、電気泳動担体を加温する工程を
加えることができる。加温する温度は、核酸試料が核酸
プローブと完全な相補性をもつ場合には解離せず、相補
性がないか又は相補性が不完全な場合には解離するよう
な温度が好ましい、この温度は、核酸試料と核酸プロー
ブの長さと塩基配列及び検出しようとする遺伝子の変異
によって種々異なるが、例えば、β−グロビン遺伝子中
のポイントミューチージョンを１９塩基長の核酸プロー
ブで検出する場合は５５°Ｃが好ましい。Furthermore, in any of the above methods, a step of heating the electrophoresis carrier can be added after the hybridization reaction is performed. The heating temperature is preferably a temperature at which the nucleic acid sample does not dissociate when it has complete complementarity with the nucleic acid probe, but dissociates when there is no complementarity or incomplete complementarity. varies depending on the length and base sequence of the nucleic acid sample and nucleic acid probe, as well as the gene mutation to be detected, but for example, when detecting a point mutation in the β-globin gene with a 19 base long nucleic acid probe, the °C is preferred.
そして、この電気泳動担体の加温により、核酸プローブ
と核酸試料とのハイブリダイゼーション反応を用いた遺
伝子変異検出法の精度を高めることができる。By heating the electrophoresis carrier, it is possible to improve the accuracy of the genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample.
上記標識核酸プローブの標識物質としては、検出可能な
ものであればいずれでもよく、３冨Ｐ等のラジオアイソ
トープでもよいが、好ましくは蛍光体又は色素或いは反
応により蛍光体又は色素を生成する酵素が用いられ７、
具体的には例えばフルオレセイン イソチアシネ−）
（ＦＩＴＣ）、エステラーゼ等が用いられる。そして、
これらの蛍光体又は色素の計測は、上記電気泳動担体中
あるいは電気泳動により上記電気泳動担体中から外に移
動せしめたものについてのいずれにおいても行うことが
できる。The labeling substance for the above-mentioned labeled nucleic acid probe may be any detectable substance, and may be a radioisotope such as 3-P, but preferably a fluorophore, a dye, or an enzyme that generates a fluorophore or dye by reaction. used7,
Specifically, for example, fluorescein, isothiacine)
(FITC), esterase, etc. are used. and,
Measurement of these fluorophores or dyes can be carried out either in the electrophoretic carrier or in those that have been moved out of the electrophoretic carrier by electrophoresis.
さらに、本発明は、上記遺伝子変異検出方法を実施する
ための遺伝子変異検出装置に係わり、核酸プローブと核
酸試料のハイブリダイゼーション反応を用いた遺伝子変
異検出装置において、核酸試料をハイブリダイゼーショ
ンさせるための核酸プローブを固定した電気泳動担体と
、上記核酸プローブを固定した電気泳動担体に正極側電
解液と負極側電解液を介して直流電圧を印加する直流電
圧印加手段と、上記電気泳動担体中又は上記正極側電解
液中の蛍光又は光の吸収を計測する計測手段とを具備し
たことを特徴とする遺伝子変異検出装置である。また、
この遺伝子変異検出装置は、計測手段が、正極側電解液
中の蛍光又は光の吸収を計測する手段である場合は、前
記計測手段によって計測される正極側電解液中の蛍光体
又は色素を濃縮するための、電解液は通過するが蛍光体
又は色素は透過しない膜を設けることができる。この膜
は上記機能を備えるものであればいずれでもよいが、例
えば石英製のポーラスガラス膜が用いられる。Furthermore, the present invention relates to a gene mutation detection device for carrying out the above gene mutation detection method, and in the gene mutation detection device using a hybridization reaction between a nucleic acid probe and a nucleic acid sample, a nucleic acid probe for hybridizing a nucleic acid sample. an electrophoretic carrier on which a probe is immobilized; a DC voltage applying means for applying a DC voltage to the electrophoretic carrier on which the nucleic acid probe is immobilized via a positive electrode side electrolyte and a negative electrode side electrolyte; The present invention is a genetic mutation detection device characterized by comprising a measuring means for measuring fluorescence or light absorption in a side electrolyte. Also,
When the measuring means is a means for measuring fluorescence or light absorption in the positive electrolyte, this genetic mutation detection device concentrates the fluorescent substance or dye in the positive electrolyte measured by the measuring means. A membrane can be provided that allows the electrolyte to pass through but not the phosphor or dye. This film may be of any material as long as it has the above-mentioned functions, but for example, a porous glass film made of quartz is used.
また、この遺伝子変異検出装置には上記電気泳動担体の
温度をコントロールするためのコントロール手段を備え
ることができる。Further, this gene mutation detection device can be equipped with a control means for controlling the temperature of the electrophoresis carrier.
さらに本発明は、上記核酸プローブと核酸試料のハイブ
リダイゼーション方法又は核酸プローブと核酸試料のハ
イブリダイゼーション反応を用いた遺伝子変異検出法に
用いる核酸プローブを固定した電気泳動担体に係るもの
である。Furthermore, the present invention relates to an electrophoresis carrier on which a nucleic acid probe is immobilized, which is used in the above-described hybridization method of a nucleic acid probe and a nucleic acid sample or a genetic mutation detection method using a hybridization reaction of a nucleic acid probe and a nucleic acid sample.
〔作 用］
電気泳動担体の上面にＤＮＡ断片試料を添加した後、２
つの電極間に直流電圧を印加して、ＤＮＡ断片試料を強
制的に担体中に移動させる。これにより、ＤＮＡ断片試
料を受動的に拡散させる場合よりも、ハイブリダイゼー
ション反応を速くできる。[Function] After adding the DNA fragment sample to the top surface of the electrophoresis carrier,
A DC voltage is applied between the two electrodes to forcibly move the DNA fragment sample into the carrier. This allows the hybridization reaction to be faster than when the DNA fragment sample is passively diffused.
また、ハイブリダイゼーション反応で結合しなかったか
又は結合が弱かったＤＮＡ断片試料を電気泳動により除
去する。これにより、溶液の注入、排出等による洗浄操
作が不要な、自動化に適した方法を実現できる。Furthermore, DNA fragment samples that did not bind or were weakly bound in the hybridization reaction are removed by electrophoresis. This makes it possible to realize a method suitable for automation that does not require cleaning operations such as injecting and discharging a solution.
さらに、ハイブリダイゼーション反応物の標識物質から
の蛍光又は光の吸収の計測は上記電気泳動担体あるいは
正極側の電解液中のいずれにおいても行うことができ、
また、後者の正極側の電解液中で計測する場合は、蛍光
体又は色素を濃縮する膜を備えることにより計測の感度
が高められる。Furthermore, measurement of fluorescence or light absorption from the labeling substance of the hybridization reaction product can be performed either in the electrophoresis carrier or in the electrolyte on the positive electrode side,
In addition, when measuring in the latter electrolyte on the positive electrode side, the sensitivity of the measurement can be increased by providing a membrane that concentrates the fluorescent substance or dye.
以下、本発明を実施例により詳細に説明する。 Hereinafter, the present invention will be explained in detail with reference to Examples.
但し、本発明はこれらの実施例により限定されるもので
ない。However, the present invention is not limited to these Examples.
実施例１ 本実施例を第１図（ａ）、 （ｂ）により説明する。Example 1 This embodiment will be explained with reference to FIGS. 1(a) and 1(b).
まず、ＤＮＡプローブを固定した電気泳動担体１は以下
のようにして調製する。ＤＮＡプローブは、ヒトβ−グ
ロビン遺伝子の５′末端から１４〜３２番目の塩基配列
と完全に相補的なりＮＡ断片（３′−ＧＡＧＧＡＣＴＣ
ＣＴＣＴＴＣＡＧＡＣＧ−５′）を、現在広く用いられ
ているフォスフォアミグイド法で合成した。ただし、合
成の最終ステップ、すなわち５′末端のグアニン（Ｇ）
を付加するステップでは、デオキシグアノシンのかわり
に５′末端にアミノ基をもつデオキジグアノシンを用い
るり、Ｍ、Ｓｍ１ｔｈ らの方法により、ＤＮＡ断片の
５′末端にアミノ基を導入した。次に、このＤＮＡプロ
ーブを高速液体クロマトグラフィー （ＨＰＬＣ）で精
製した後、２．５％アクロレイン水溶液に加えて水冷下
３０分間反応させた。これをＰＢＳ緩衝液でよく透析し
た後、さらに５％アクリルアミド−Ｎ、 Ｎ’ −メチ
レノビスアクリル−アミトン容液（アクリルアミド：Ｎ
、 Ｎ−メチレンビスアクリルアミド＝２０：１）、最
終濃度０．０８％のＮ、 Ｎ、 Ｎ’、 Ｎ−テトら
メチルエチレンジアミン、最終濃度０．１％の過硫酸ア
ンモニウムを加えてガラス管２に注入し、ゲル化させて
電気泳動担体１とした。First, electrophoresis carrier 1 on which DNA probes are immobilized is prepared as follows. The DNA probe was an NA fragment (3'-GAGGACTC) that was completely complementary to the 14th to 32nd base sequence from the 5' end of the human β-globin gene.
CTCTTCAGACG-5') was synthesized by the currently widely used phosphoramid method. However, the final step of synthesis, i.e., guanine (G) at the 5' end,
In the step of adding , an amino group was introduced into the 5' end of the DNA fragment by using deoxyguanosine having an amino group at the 5' end instead of deoxyguanosine or by the method of M. Smlth et al. Next, this DNA probe was purified by high performance liquid chromatography (HPLC), and then added to a 2.5% acrolein aqueous solution and reacted for 30 minutes under water cooling. After thoroughly dialyzing this with PBS buffer, 5% acrylamide-N,N'-methylenobisacryl-amiton solution (acrylamide:N
, N-methylenebisacrylamide = 20:1), N, N, N', N-tetramethylethylenediamine with a final concentration of 0.08%, ammonium persulfate with a final concentration of 0.1% and injected into glass tube 2. The gel was formed into electrophoresis carrier 1.
ＤＮＡ断片試料としては、変異を含まない正常人のβ−
グロビン遺伝子（βＡ）と５′末端から２０番目のアデ
ノシン（Ａ）がチミン（Ｔ）に変異した（ポイントミュ
ーチージョン）、鎌状赤血球貧血症患者のβ−グロビン
遺伝子（β３）を制限酵素ＢａｍＨ１で切断したもの（
β−グロビン遺伝子の５′末端付近を含む、長さ約１８
００塩基対の断片）を使用した。As a DNA fragment sample, normal human β-
The globin gene (βA) and the 20th adenosine (A) from the 5' end are mutated to thymine (T) (point mutation), and the β-globin gene (β3) of a patient with sickle cell anemia is converted using the restriction enzyme BamH1. The one cut with (
Approximately 18 in length, including near the 5' end of the β-globin gene
00 base pair fragment) was used.
上記ＤＮＡ断片試料を加熱変性させて一本ｕｉＤＮＡと
してから、温度コントローラ３によって４５℃に保たれ
ているＤＮＡプローブを固定した電気泳動担体１の上端
に注入し、上部電解液槽４中の負極６と下部電解液槽７
中の正極９の間に直流電源１０で電圧を印加した。これ
により、ＤＮＡ断片試料は電気泳動担体ｌの中へ強制的
に電気泳動されるため、電気泳動を行なわずに受動的に
拡散させる場合に比べて、ハイブリダイゼーション反応
を速く進めることができる。After heat-denaturing the DNA fragment sample to obtain a single uiDNA, it is injected onto the upper end of the electrophoresis carrier 1 on which the DNA probe immobilized, which is maintained at 45°C by the temperature controller 3, is placed on the negative electrode 6 in the upper electrolyte tank 4. and lower electrolyte tank 7
A voltage was applied between the positive electrodes 9 inside with a DC power supply 10. As a result, the DNA fragment sample is forcibly electrophoresed into the electrophoresis carrier 1, so that the hybridization reaction can proceed faster than when it is passively diffused without electrophoresis.
次に、電気泳動担体ｌの温度を温度コントローラ３によ
って５５°Ｃに変更してから、再び２つの電極６，９の
間に電圧を印加し、ＤＮＡプローブと完全な相補性を持
たないために解離したＤＮＡ断片試料を電気泳動により
除去した。Next, the temperature of the electrophoresis carrier l is changed to 55°C by the temperature controller 3, and then a voltage is applied between the two electrodes 6 and 9 again to avoid complete complementarity with the DNA probe. Dissociated DNA fragment samples were removed by electrophoresis.
さらに、電気泳動担体の温度を４５°Ｃにもどしてから
、エステラーゼで標識した第二のＤＮＡプローブを電気
泳動担体１の上端に注入し、電気泳動した。このＤＮＡ
プローブ（標識プローブ）は、電気泳動担体１に固定し
たプローブ（固定化プローブ）と同様にフォスフォアミ
グイド法で合成したＤＮＡ断片（３’−ＣＣＡＣＴＴＧ
ＣＡＣＣＴＡＣＴＴＣＡＡＣ−５’）の５′末端をエス
テラーゼで標識したものであるが、固定化プローブとは
異なる部位、すなわちβ−グロビン遺伝子の５′末端か
ら５３〜７２番目の塩基配列に相補的である。したがっ
て、ＤＮＡ断片試料が固定化プローブに結合して電気泳
動担体１中に残っていれば、標識プローブもＤＮＡ断片
試料の別の部位に結合して電気泳動担体１中に残るが、
ＤＮＡ断片試料が残っていなければ、標識プローブは電
気泳動担体１中に残らず通過する。Furthermore, after returning the temperature of the electrophoresis carrier to 45° C., a second DNA probe labeled with esterase was injected into the upper end of the electrophoresis carrier 1, and electrophoresis was performed. this DNA
The probe (labeled probe) is a DNA fragment (3'-CCACTTG
Although the 5' end of CACCTACTTCAAC-5') is labeled with esterase, it is complementary to a different site from that of the immobilized probe, that is, the 53rd to 72nd base sequence from the 5' end of the β-globin gene. Therefore, if the DNA fragment sample binds to the immobilized probe and remains in the electrophoresis carrier 1, the labeled probe also binds to another site of the DNA fragment sample and remains in the electrophoresis carrier 1;
If no DNA fragment sample remains, all labeled probes pass through the electrophoresis carrier 1.
最後に、標識酵素エステラーゼの基質であるＦＤＡ （
フルオレセインジアセテート）を同様に電気泳動担体ｌ
の上端に注入して電気泳動した後、酵素反応で生じた蛍
光物質フルオレセインの蛍光を、電気泳動担体１中で測
定した。Finally, FDA (
Fluorescein diacetate) was similarly used as an electrophoresis carrier.
After injecting it into the upper end of the electrophoresis carrier 1 and performing electrophoresis, the fluorescence of the fluorescent substance fluorescein produced by the enzyme reaction was measured in the electrophoresis carrier 1.
キセノンランプの光源１１から出た光を干渉フィルター
１２に通して４９０ｎｍの波長の光を選択した後、レン
ズ１３で集光して電気泳動担体１に励起光を照射した。Light emitted from a light source 11 of a xenon lamp was passed through an interference filter 12 to select light with a wavelength of 490 nm, and then focused by a lens 13 to irradiate the electrophoresis carrier 1 with excitation light.
励起光に対して９０°の方向から、レンズ１７、カット
オフフィルター１８、干渉フィルター１９を通して、５
１０ｎｍ近傍の波長の光を選択的にフォトマル２０で検
出した。なお、入射窓１４の反対側に窓１６を設け、電
気泳動担体１を通過した励起光を外部に導くことにより
、散乱光の影響を少なくした。フォトマル２０の出力は
増幅器２１で増幅した後、レコーダ２２で記録した。5 through the lens 17, the cut-off filter 18, and the interference filter 19 from a direction of 90° to the excitation light.
Light with a wavelength around 10 nm was selectively detected using Photomar 20. Note that a window 16 was provided on the opposite side of the entrance window 14 to guide the excitation light that passed through the electrophoretic carrier 1 to the outside, thereby reducing the influence of scattered light. The output of the photomultiplier 20 was amplified by an amplifier 21 and then recorded by a recorder 22.
測定の結果、ＤＮＡ断片試料が変異を含まない正常人の
β−グロビン遺伝子（βＡ）の断片で、固定化ＤＮＡプ
ローブと完全な相補性をもつ場合には、蛍光が検出され
たが、ＤＮＡ断片試料が変異（ポイントミューチージョ
ン）を含む鎌状赤血球貧血症患者のβグロビン遺伝子（
βＳ）の断片で、固定化ＤＮＡプローブと１塩基だけ相
補性をもたない場合には、蛍光は検出されなかった。確
認のために、固定化ＤＮＡプローブをβ３遺伝子に完全
な相補性をもつもの（３′−ＧＡＧＧＡＣＡＣＣＴＣＴ
ＴＣＡＧＡＣＧ−５′）にかえて同様の測定を行なった
ところ、ＤＮＡ断片試料がβ１遺伝子の断片の場合には
蛍光が検出されず、β３遺伝子の断片の場合には蛍光が
検出された。このように、変異を含む遺伝子断片と含ま
ない遺伝子断片を、蛍光が検出されるか否かによって区
別できるため、β−グロビン遺伝子断片中の変異（ポイ
ントミューチージョン）を検出することができた。As a result of the measurement, fluorescence was detected when the DNA fragment sample was a fragment of the β-globin gene (βA) of a normal person without mutations and had complete complementarity with the immobilized DNA probe; The β-globin gene of a sickle cell anemia patient whose sample contains a mutation (point mutation)
No fluorescence was detected in the case where the fragment of βS) had no complementarity with the immobilized DNA probe by just one base. For confirmation, we used an immobilized DNA probe with complete complementarity to the β3 gene (3'-GAGGACACCTCT).
When similar measurements were carried out using TCAGACG-5'), no fluorescence was detected when the DNA fragment sample was a β1 gene fragment, but fluorescence was detected when the DNA fragment sample was a β3 gene fragment. In this way, gene fragments that contain mutations and those that do not can be distinguished by whether or not fluorescence is detected, making it possible to detect mutations (point mutations) in β-globin gene fragments. .
なお、本実施例では標識物質として酵素（エステラーゼ
）を用い、酵素反応によって生成するＦＤＡの蛍光を測
定したが、標識物質としてＦＩＴＣ等の蛍光物質を用い
、酵素や酵素反応を用いずに、直接その蛍光を測定して
もよい。In this example, an enzyme (esterase) was used as a labeling substance and the fluorescence of FDA generated by the enzymatic reaction was measured. The fluorescence may be measured.
以上のように、本実施例により、高速で自動化容易な遺
伝子変異検出法及び装置を実現できた。As described above, according to this example, a method and apparatus for detecting genetic mutations that are fast and easy to automate were realized.
実施例２ 次に、第２の実施例を第２図により説明する。Example 2 Next, a second embodiment will be explained with reference to FIG.
本実施例と実施例１の違いは、蛍光物質フルオレセイン
の蛍光を、電気泳動担体１中ではなく、下部電解液８中
で測定するところにある。前記実施例の最後のステップ
で、ＦＤＡを電気泳動により電気泳動担体１中に移動さ
せた後、さらに電気泳動を続けて酵素反応で生じた蛍光
物質フルオレセインを下部電解液８中に泳動させた。そ
して、フルオレセインの蛍光を第２図に示す装置を用い
て、下部電解液中で測定した。The difference between this example and Example 1 is that the fluorescence of the fluorescent substance fluorescein is measured not in the electrophoresis carrier 1 but in the lower electrolyte 8. In the last step of the above example, after FDA was transferred into the electrophoretic carrier 1 by electrophoresis, electrophoresis was continued to transfer the fluorescent substance fluorescein produced by the enzyme reaction into the lower electrolytic solution 8. Then, the fluorescence of fluorescein was measured in the lower electrolyte using the apparatus shown in FIG.
本実施例によれば、前記実施例と同様の効果に加えて、
散乱光と妨害蛍光の大きい電気泳動担体中ではなく、こ
れらの小さい電解液中でフルオレセインの蛍光を測定す
るので、高感度な蛍光測定が可能であるという効果があ
る。According to this embodiment, in addition to the same effects as in the previous embodiment,
Since the fluorescence of fluorescein is measured in these small electrolytes rather than in the electrophoretic carrier, which has a large amount of scattered light and interfering fluorescence, there is an advantage that highly sensitive fluorescence measurement is possible.
実施例３ 次に、第３の実施例を第３図により説明する。Example 3 Next, a third embodiment will be explained with reference to FIG.
本実施例と実施例２の違いは、電気泳動担体１の下端と
下部電解液８の間にアクリル製の膜保持具２３に取り付
けたポーラスガラス膜２４を配置することにより、小容
積の電解液槽２５を構成した点にある。上記ポーラスガ
ラス膜２４は、ゾルゲル法でテトラメトキシシランをメ
タノールと水溶媒中で反応させたもので、電解液中の電
解質は透過させるが、蛍光体は透過させないという性質
をもつ石英ガラスである。したがって、酵素反応によっ
て生成した蛍光体ＦＤＡは小容積の電解液槽２５中に濃
縮される。本実施例では、上記過程によって濃縮された
蛍光体を含む電解液をガイド穴２６を通してピペット２
７を用いて蛍光セル２８に導いた。ピペット２７は回転
上下機構２９に保持した。蛍光セル２８中の蛍光体は、
第２図に示したのと同様な光学系で蛍光計測した。The difference between this example and Example 2 is that a porous glass membrane 24 attached to an acrylic membrane holder 23 is placed between the lower end of the electrophoretic carrier 1 and the lower electrolyte 8, so that a small volume of electrolyte can be obtained. This is due to the structure of the tank 25. The porous glass membrane 24 is made by reacting tetramethoxysilane with methanol in a water solvent using a sol-gel method, and is a quartz glass that allows the electrolyte in the electrolytic solution to pass through, but not the phosphor. Therefore, the phosphor FDA generated by the enzymatic reaction is concentrated in the small volume electrolyte tank 25. In this embodiment, the electrolyte containing the phosphor concentrated through the above process is passed through the guide hole 26 to the pipette 2.
7 to the fluorescent cell 28. The pipette 27 was held by a rotating up/down mechanism 29. The phosphor in the fluorescent cell 28 is
Fluorescence measurements were performed using an optical system similar to that shown in FIG.
本実施例によれば、実施例２と同様の効果に加えて、蛍
光物質ＦＤＡを小容積の電解液中に濃縮できるため、さ
らに高感度な蛍光測定が可能であるという効果がある。According to the present example, in addition to the same effects as in Example 2, the fluorescent substance FDA can be concentrated in a small volume of electrolyte, so that fluorescence measurement with even higher sensitivity is possible.
本発明によれば、ＤＮＡ断片試料を電気泳動により強制
的に電気泳動担体中に移動させるので、従来のニトロセ
ルロース膜で用いた方法で受動的に拡散させる場合より
も、ハイブリダイゼーション反応を速くでき、短時間で
完了できる。また、ハイブリダイゼーション反応で結合
しなかったか又は結合が弱かったＤＮＡ試料を、溶液の
注入、排出等による洗浄操作を用いずに、電気泳動によ
って容易に除去することができる。したがって、本発明
によれば高速で自動化容易な遺伝子変異検出方法を実現
できる。更に本発明は、標識物質の蛍光体又は色素を濃
縮することにより計測感度を高めることができる。According to the present invention, since the DNA fragment sample is forcibly moved into the electrophoretic carrier by electrophoresis, the hybridization reaction can be performed faster than when the DNA fragment sample is passively diffused by the method used with conventional nitrocellulose membranes. , can be completed in a short time. Furthermore, DNA samples that did not bind or were weakly bound in the hybridization reaction can be easily removed by electrophoresis without using washing operations such as injection and discharge of solutions. Therefore, according to the present invention, a method for detecting genetic mutations that is fast and easy to automate can be realized. Furthermore, the present invention can increase measurement sensitivity by concentrating the fluorescent substance or dye of the labeling substance.
第１図（ａ）、 （ｂ）は各々本発明の第一の実施例で
用いた装置の縦断面図と横断面図、第２図は本発明の第
二の実施例で用いた装置の縦断面図、第３図は本発明の
第三の実施例で用いた装置の縦断面図の一部拡大図であ
る。
１・・・電気泳動担体、２・・・ガラス管、３・・・温
度コントローラ、４・・・上部電解液槽、５・・・上部
（負極側）電解液、６・・・負極、７・・・下部（正極
側）電解液槽、８・・・下部（正極側）電解液、９・・
・正極、１０・・・直流電源、１１・・・光源、１２．
１９・・・干渉フィルター１３、１７・・・レンズ、Ｉ
４・・・入射窓、Ｉ５・・・検出窓、１６・・・窓、１
８・・・カットオフフィルター、２０・・・フォトマル
、２１・・・増幅器、２２・・・レコーダー、２３・・
・膜保持具、２４・・・ポーラスガラス膜、２５・・・
小容積の電解液槽、２６・・・ガイド穴、２７・・・ピ
ペット、２８・・・蛍光セル、２９・・・回転上下機構
。
第２図
第６図FIGS. 1(a) and (b) are longitudinal and cross-sectional views, respectively, of the apparatus used in the first embodiment of the present invention, and FIG. 2 is a longitudinal sectional view and cross-sectional view of the apparatus used in the second embodiment of the present invention. FIG. 3 is a partially enlarged longitudinal sectional view of the apparatus used in the third embodiment of the present invention. DESCRIPTION OF SYMBOLS 1... Electrophoresis carrier, 2... Glass tube, 3... Temperature controller, 4... Upper electrolyte tank, 5... Upper (negative electrode side) electrolyte solution, 6... Negative electrode, 7 ...Lower (positive electrode side) electrolyte tank, 8...Lower (positive electrode side) electrolyte solution, 9...
- Positive electrode, 10... DC power supply, 11... Light source, 12.
19...Interference filter 13, 17...Lens, I
4...Incidence window, I5...Detection window, 16...Window, 1
8... Cutoff filter, 20... Photomultiple, 21... Amplifier, 22... Recorder, 23...
・Membrane holder, 24... Porous glass membrane, 25...
Small volume electrolyte tank, 26... Guide hole, 27... Pipette, 28... Fluorescent cell, 29... Rotating up and down mechanism. Figure 2 Figure 6
Claims (1)
方法において、核酸プローブを電気泳動担体中に固定し
、核酸試料を電気泳動によって電気担体中に移動せしめ
ることを特徴とする核酸試料のハイブリダイゼーション
方法。 ２、核酸プローブと核酸試料のハイブリダイゼーション
反応を用いた遺伝子変異検出法において、核酸プローブ
を電気泳動担体中に固定し、核酸試料を電気泳動によっ
て電気泳動担体中に移動せしめてハイブリダイゼーショ
ン反応を行なわせ、上記核酸プローブと結合しなかった
上記核酸試料を電気泳動によって移動せしめて上記電気
泳動担体中から除去することを特徴とする遺伝子変異検
出方法。 ３、核酸プローブと核酸試料のハイブリダイゼーション
反応を用いた遺伝子変異検出法において、核酸プローブ
を電気泳動担体中に固定し、核酸試料を電気泳動によっ
て電気泳動担体中に移動せしめてハイブリダイゼーショ
ン反応を行わせ、次いで前記電気泳動担体を加温した後
、上記核酸プローブと結合しなかった上記核酸試料を電
気泳動によって移動せしめて上記電気泳動担体中から除
去することを特徴とする遺伝子変異検出方法。 ４、核酸プローブと核酸試料のハイブリダイゼーション
反応を用いた遺伝子変異検出法において、核酸プローブ
を電気泳動担体中に固定し、核酸試料を電気泳動によっ
て電気泳動担体中に移動せしめてハイブリダイゼーショ
ン反応を行なわせ、上記核酸プローブと結合しなかった
上記核酸試料を電気泳動によって移動せしめて電気泳動
担体中から除去し、さらに標識核酸プローブを電気泳動
によって電気泳動担体中に移動せしめてハイブリダイゼ
ーション反応を行なわせ、上記核酸プローブと結合しな
かった上記標識核酸プローブを電気泳動によって移動せ
しめて電気泳動担体中から除去した後、上記核酸試料と
結合した標識核酸プローブの標識を検出することを特徴
とする遺伝子変異検出方法。 ５、核酸プローブと核酸試料のハイブリダイゼーション
反応を用いた遺伝子変異検出法において、核酸プローブ
を電気泳動担体中に固定し、核酸試料を電気泳動によっ
て電気泳動担体中に移動せしめてハイブリダイゼーショ
ン反応を行わせ、次いで前記電気泳動担体を加温した後
、上記核酸プローブと結合しなかった上記核酸試料を電
気泳動によって移動せしめて上記電気泳動担体中から除
去し、さらに標識核酸プローブを電気泳動によって電気
泳動担体中に移動せしめてハイブリダイゼーション反応
を行わせ、次いで上記電気泳動担体を加温した後、上記
核酸プローブと結合しなかった上記標識核酸プローブを
電気泳動によって移動せしめて電気泳動担体中から除去
した後、上記核酸試料と結合した標識核酸プローブの標
識を検出することを特徴とする遺伝子変異検出方法。 ６、標識は蛍光体又は色素であり、これらを電気泳動担
体中で検出することを特徴とする請求項４又は５記載の
遺伝子変異検出方法。 ７、標識は酵素であり、当該酵素による酵素反応によっ
て生成する蛍光体又は色素を電気泳動担体中で検出する
か、あるいは電気泳動によって上記電気泳動担体中から
外に移動せしめて検出することを特徴とする請求項４又
は５記載の遺伝子変異検出方法。 ８、核酸プローブと核酸試料のハイブリダイゼーション
反応を用いた遺伝子変異検出装置において、核酸試料を
ハイブリダイゼーションさせるための核酸プローブを固
定した電気泳動担体と、上記核酸プローブを固定した電
気泳動担体に正極側電解液と負極側電解液を介して直流
電圧を印加する直流電圧印加手段と、上記電気泳動担体
中又は上記正極側電解液中の蛍光又は光の吸収を計測す
る計測手段とを具備したことを特徴とする遺伝子変異検
出装置。 ９、計測手段が正極側電解液中の蛍光又は光の吸収を計
測する手段であって、前記計測手段によって計測される
正極側電解液中の蛍光体又は色素を濃縮するための、電
解液は通過するが蛍光体又は色素は透過しない膜を設け
たことを特徴とする請求項８記載の遺伝子変異検出装置
。 １０、上記電気泳動担体の温度をコントロールする手段
を具備したことを特徴とする請求項８又は９記載の遺伝
子変異検出装置。 １１、核酸プローブと核酸試料のハイブリダイゼーショ
ン方法又は核酸プローブと核酸試料のハイブリダイゼー
ション反応を用いた遺伝子変異検出法に用いる核酸プロ
ーブを固定した電気泳動担体。[Claims] 1. A method for hybridizing a nucleic acid probe and a nucleic acid sample, which is characterized in that the nucleic acid probe is immobilized in an electrophoretic carrier, and the nucleic acid sample is moved into the electrophoretic carrier by electrophoresis. Hybridization method. 2. In a genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample, the nucleic acid probe is immobilized in an electrophoresis carrier, and the nucleic acid sample is moved into the electrophoresis carrier by electrophoresis to perform a hybridization reaction. A method for detecting genetic mutations, characterized in that the nucleic acid sample that does not bind to the nucleic acid probe is moved by electrophoresis and removed from the electrophoresis carrier. 3. In a genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample, the nucleic acid probe is immobilized in an electrophoretic carrier, and the nucleic acid sample is moved into the electrophoretic carrier by electrophoresis to perform a hybridization reaction. A method for detecting gene mutations, comprising: heating the electrophoretic carrier, and then moving the nucleic acid sample that did not bind to the nucleic acid probe by electrophoresis to remove it from the electrophoretic carrier. 4. In a genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample, the nucleic acid probe is immobilized in an electrophoretic carrier, and the nucleic acid sample is moved into the electrophoretic carrier by electrophoresis to perform a hybridization reaction. The nucleic acid sample that did not bind to the nucleic acid probe is moved by electrophoresis to be removed from the electrophoresis carrier, and the labeled nucleic acid probe is further moved into the electrophoresis carrier by electrophoresis to perform a hybridization reaction. , a gene mutation characterized in that the labeled nucleic acid probe that has not bound to the nucleic acid probe is moved by electrophoresis and removed from the electrophoresis carrier, and then the label of the labeled nucleic acid probe that has bound to the nucleic acid sample is detected. Detection method. 5. In a genetic mutation detection method using a hybridization reaction between a nucleic acid probe and a nucleic acid sample, the nucleic acid probe is immobilized in an electrophoretic carrier, and the nucleic acid sample is moved into the electrophoretic carrier by electrophoresis to perform a hybridization reaction. Then, after heating the electrophoresis carrier, the nucleic acid sample that did not bind to the nucleic acid probe is moved by electrophoresis to be removed from the electrophoresis carrier, and the labeled nucleic acid probe is further moved by electrophoresis. The labeled nucleic acid probe that did not bind to the nucleic acid probe was moved by electrophoresis and removed from the electrophoresis carrier after the electrophoresis carrier was heated. and then detecting the label of the labeled nucleic acid probe bound to the nucleic acid sample. 6. The method for detecting genetic mutations according to claim 4 or 5, characterized in that the label is a fluorescent substance or a dye, and these are detected in an electrophoresis carrier. 7. The label is an enzyme, and the fluorescent substance or dye produced by the enzyme reaction is detected in the electrophoretic carrier, or is detected by being moved out of the electrophoretic carrier by electrophoresis. The method for detecting genetic mutations according to claim 4 or 5. 8. In a genetic mutation detection device that uses a hybridization reaction between a nucleic acid probe and a nucleic acid sample, an electrophoresis carrier on which a nucleic acid probe is immobilized for hybridizing a nucleic acid sample, and a positive electrode side on the electrophoresis carrier on which the nucleic acid probe is immobilized. A DC voltage applying means for applying a DC voltage through the electrolyte and the negative electrolyte; and a measuring means for measuring fluorescence or light absorption in the electrophoretic carrier or the positive electrolyte. Characteristic gene mutation detection device. 9. The measuring means is a means for measuring fluorescence or light absorption in the positive electrolyte, and the electrolytic solution is for concentrating the phosphor or dye in the positive electrolyte measured by the measuring means. 9. The gene mutation detection device according to claim 8, further comprising a membrane through which fluorescent material or dye passes through but not through which fluorescent substance or dye passes. 10. The gene mutation detection device according to claim 8 or 9, further comprising means for controlling the temperature of the electrophoresis carrier. 11. An electrophoresis carrier on which a nucleic acid probe is immobilized for use in a method of hybridizing a nucleic acid probe and a nucleic acid sample or a method of detecting genetic mutations using a hybridization reaction of a nucleic acid probe and a nucleic acid sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1178933A JPH0347097A (en) | 1989-07-13 | 1989-07-13 | Hybridization method, gene mutation detection using same and apparatus therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1178933A JPH0347097A (en) | 1989-07-13 | 1989-07-13 | Hybridization method, gene mutation detection using same and apparatus therefor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0347097A true JPH0347097A (en) | 1991-02-28 |
Family
ID=16057180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1178933A Pending JPH0347097A (en) | 1989-07-13 | 1989-07-13 | Hybridization method, gene mutation detection using same and apparatus therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0347097A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993024625A1 (en) * | 1992-05-28 | 1993-12-09 | Morinaga Milk Industry Co., Ltd. | Gene macromolecule reaction method and apparatus therefor |
| WO1998051823A1 (en) * | 1997-05-16 | 1998-11-19 | Mosaic Technologies | Electrophoretic analysis of molecules using immobilized probes |
| US6214187B1 (en) | 1998-06-18 | 2001-04-10 | Mosaic Technologies | Denaturing gradient affinity electrophoresis and methods of use thereof |
| US6238927B1 (en) | 1998-10-05 | 2001-05-29 | Mosaic Technologies, Incorporated | Reverse displacement assay for detection of nucleic acid sequences |
| US6255051B1 (en) | 1997-11-06 | 2001-07-03 | Mosaic Technologies Inc. | Multiple sequential polynucleotide displacement reactions for signal amplification and processing |
| WO2000060120A3 (en) * | 1999-04-02 | 2001-09-13 | Mosaic Technologies | Methods of detecting microorganism using immobilized probes |
| WO2000060118A3 (en) * | 1999-04-02 | 2002-07-11 | Mosaic Technologies Inc | Electrophoretic analysis of target molecules using adapter molecules |
| JP2002537781A (en) * | 1999-02-26 | 2002-11-12 | モザイク テクノロジーズ | Biochemical purification device with immobilized capture probe and its use |
| US6692912B1 (en) | 1997-03-05 | 2004-02-17 | Matrix Technologies Corporation | Nucleic acid-containing polymerizable complex |
| WO2012108499A1 (en) | 2011-02-10 | 2012-08-16 | 三菱レイヨン株式会社 | Method for detecting nucleic acid |
-
1989
- 1989-07-13 JP JP1178933A patent/JPH0347097A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993024625A1 (en) * | 1992-05-28 | 1993-12-09 | Morinaga Milk Industry Co., Ltd. | Gene macromolecule reaction method and apparatus therefor |
| US6692912B1 (en) | 1997-03-05 | 2004-02-17 | Matrix Technologies Corporation | Nucleic acid-containing polymerizable complex |
| WO1998051823A1 (en) * | 1997-05-16 | 1998-11-19 | Mosaic Technologies | Electrophoretic analysis of molecules using immobilized probes |
| AU730491B2 (en) * | 1997-05-16 | 2001-03-08 | Exact Sciences Corporation | Electrophoretic analysis of molecules using immobilized probes |
| US6255051B1 (en) | 1997-11-06 | 2001-07-03 | Mosaic Technologies Inc. | Multiple sequential polynucleotide displacement reactions for signal amplification and processing |
| US6214187B1 (en) | 1998-06-18 | 2001-04-10 | Mosaic Technologies | Denaturing gradient affinity electrophoresis and methods of use thereof |
| US6238927B1 (en) | 1998-10-05 | 2001-05-29 | Mosaic Technologies, Incorporated | Reverse displacement assay for detection of nucleic acid sequences |
| JP2002537781A (en) * | 1999-02-26 | 2002-11-12 | モザイク テクノロジーズ | Biochemical purification device with immobilized capture probe and its use |
| WO2000060120A3 (en) * | 1999-04-02 | 2001-09-13 | Mosaic Technologies | Methods of detecting microorganism using immobilized probes |
| WO2000060118A3 (en) * | 1999-04-02 | 2002-07-11 | Mosaic Technologies Inc | Electrophoretic analysis of target molecules using adapter molecules |
| WO2012108499A1 (en) | 2011-02-10 | 2012-08-16 | 三菱レイヨン株式会社 | Method for detecting nucleic acid |
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