JPH03243858A - Detection of dna - Google Patents
Detection of dnaInfo
- Publication number
- JPH03243858A JPH03243858A JP2040303A JP4030390A JPH03243858A JP H03243858 A JPH03243858 A JP H03243858A JP 2040303 A JP2040303 A JP 2040303A JP 4030390 A JP4030390 A JP 4030390A JP H03243858 A JPH03243858 A JP H03243858A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- electrophoresis
- polymer
- capillary
- amplified
- 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.)
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Abstract
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明はＤＮＡの検出方法に関するものである。[Detailed description of the invention] [Industrial application field] The present invention relates to a method for detecting DNA.
更に詳しくは、遺伝子増幅技術とキャピラリー電気泳動
法を利用したＤＮＡの検出方法に関する。More specifically, the present invention relates to a DNA detection method using gene amplification technology and capillary electrophoresis.
［従来の技術］
近年、遺伝子工学の発展に伴いＤＮＡの検出方法が開発
され試みられるようになった。例えば、オリゴヌクレオ
チドを用いたＤＮＡプローブ法、ハイブリダイゼーショ
ン法が試みられ、またＤＮＡをエチジウムブロマイドを
含有するアガロースゲルを用いて電気泳動を行い分離し
、ＵＶを照射して発生する蛍光を写真に撮るという方法
でＤＮＡの検出が行われている。[Prior Art] In recent years, with the development of genetic engineering, DNA detection methods have been developed and attempted. For example, DNA probe methods and hybridization methods using oligonucleotides have been attempted, and DNA is separated by electrophoresis using an agarose gel containing ethidium bromide, and the fluorescence generated by UV irradiation is photographed. DNA is detected using this method.
このようなりＮＡの検出を利用した応用は、種々の分野
に広がっており、例えば、遺伝子導入された形質転換体
の検出、遺伝病の診断等が挙げられるが、微生物検査に
おける応用もその１つである。Applications using NA detection are spreading to various fields, such as detection of gene-introduced transformants and diagnosis of genetic diseases, but one example is application in microbial testing. It is.
微生物検査は、従来、感染症の診断あるいは食品の細菌
検査においてまずその試料中に存在する細菌等を好気条
件及び嫌気条件下等において増菌培養を行い集落の観察
を行った後、選択培地を用いて分離培養し集落の観察を
行い菌の諸性状の検査を行うことにより菌の同定を行っ
ている。あるいは、増菌培養の段階を省き試料中に存在
する細菌等を好気条件及び嫌気条件下等において選択培
地を用いて分離培養し集落の観察を行い菌の諸性状の検
査を行うことにより菌の同定を行っている（臨床検査技
術全書第７巻微生物検査、小酒井望編集、１９８５年医
学書院発行）。Conventionally, in the diagnosis of infectious diseases or bacterial testing of food, microbial testing first involves enlarging the bacteria present in the sample under aerobic and anaerobic conditions, observing the colonies, and then using a selective medium. Bacteria are identified by isolating and culturing them, observing the colonies, and testing various properties of the bacteria. Alternatively, the bacteria present in the sample can be isolated and cultured using a selective medium under aerobic or anaerobic conditions, and the colonies can be observed and various properties of the bacteria can be tested. (Clinical Testing Techniques Complete Book Volume 7 Microbial Testing, edited by Nozomi Kosakai, published by Igaku Shoin in 1985).
近年、このような微生物検査においてもオリゴヌクレオ
チドを用いたＤＮＡプローブ法あるいはハイブリダイゼ
ーション法が試みられるようになってきており、ＤＮＡ
の検出による微生物検査の実用化が期待されている。In recent years, DNA probe methods or hybridization methods using oligonucleotides have been attempted in such microbial testing, and DNA
It is expected that microbial testing will be put to practical use by detecting .
また、ＤＮＡはオリゴヌクレオチドをブライマーとして
用いた遺伝子増幅法（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉ
ｎＲｅａｃｔｉｏｎ法以下、略してＰＣＲ法Ｈ５ａｉｋ
ｉら、５ｃｉｅｎｃｅ、 ２３０．１３５０ （１９８
５）によって増幅させる方法が知られており、ＤＮＡの
検出には増幅させたＤＮＡを用いる方法が試みられてい
る。In addition, DNA can be processed using a gene amplification method (Polymerase Chai) using oligonucleotides as primers.
nReaction method, abbreviated as PCR method H5aik
i et al., 5science, 230.1350 (198
5) is known, and methods using amplified DNA have been attempted for DNA detection.
［発明が解決しようとする課題］
ＤＮＡの検出方法のうち、特に微生物検査等においては
、オリゴヌクレオチドを標識修飾したプローブにより膜
上あるいは他の支持体上でハイブリダイゼーションを行
い、標識修飾したプローブを検出する方法は、十分な検
出感度と選択性を得るのが難しいのが実情である。[Problems to be Solved by the Invention] Among DNA detection methods, particularly in microbial testing, oligonucleotides are hybridized on membranes or other supports using probes modified with labels, and the probes modified with labels are hybridized. The reality is that it is difficult to obtain sufficient detection sensitivity and selectivity using detection methods.
また、検体から得られた微生物由来のＤＮＡをＰＣＲ法
によって増幅し、その増幅したＤＮＡをアガロースゲル
電気泳動によって検出する方法では、ゲル作製に手間が
かかる、ゲルが扱いにくい、サンプルをゲルにアプライ
するのに手間がかかる、見た目には分かりやすいが定量
性がよくないなどの短所がある。従って、特に微生物検
査のような迅速性、簡便性かつ高感度が要求される分野
では自動化あるいは装置化しようとした場合に問題とな
っている。このような状況下、迅速、簡便かつ検出感度
の高いＤＮＡの検出方法の開発が期待されている。In addition, the method of amplifying DNA derived from microorganisms obtained from a sample by PCR and detecting the amplified DNA by agarose gel electrophoresis has the disadvantages that gel preparation is time-consuming, gels are difficult to handle, and the sample is not applied to the gel. It has disadvantages such as being time-consuming and visually easy to understand, but not very quantitative. Therefore, this poses a problem when attempting to automate or implement equipment, especially in fields such as microbial testing that require rapidity, simplicity, and high sensitivity. Under these circumstances, the development of a DNA detection method that is quick, simple, and has high detection sensitivity is expected.
本発明の目的は、まさにこの点にあり、ＰＣＲ法を利用
して増幅されたＤＮＡの検出を迅速かつ簡便に行う方法
を提供することにある。The purpose of the present invention is precisely in this respect, and is to provide a method for quickly and easily detecting DNA amplified using the PCR method.
詳しくは、理論段数が高く十分な分離が得られる、分析
時間が短く時間当りの試料処理能力が高い、サンプルの
インジェク、トが行いやすい、サンプルが少なくてよく
、ゲルの作製が不要である、またこれらのことよりラン
ニングコストを低く抑えることができる、その他定量性
および再現性がよい等の長所を持つＤＮＡの検出方法を
提供することにある。In detail, it has a high number of theoretical plates and provides sufficient separation, short analysis times and high sample throughput per hour, easy sample injection, small sample size, and no need for gel preparation. Furthermore, it is an object of the present invention to provide a DNA detection method which has advantages such as being able to keep running costs low and having good quantitative performance and reproducibility.
［課題を解決するための手段および作用］本発明者は、
上記の目的を達成するため鋭意検討を行った結果、微生
物検査等を行う際に検体中のＤＮＡの一部分をまずＰＣ
Ｒ法を利用して増幅し、増幅したＤＮＡの検出をキャピ
ラリー電気泳動法を用いて行うことにより、迅速、簡便
かつ高感度にＤＮＡを検出することができることを見出
し、さらに研究を重ねて本発明を完成するに至った。[Means and effects for solving the problem] The present inventors:
As a result of intensive research to achieve the above objectives, we have found that when performing microbiological tests, etc., a portion of the DNA in the specimen is first
It was discovered that by amplifying using the R method and detecting the amplified DNA using capillary electrophoresis, DNA can be detected quickly, easily, and with high sensitivity.After further research, the present invention was developed. I was able to complete it.
ここで、本発明で検出されるＤＮＡは、ＤＮＡの検出の
目的によりどのような由来のものであってもよい。例え
ば、微生物検査においては、微生物由来のＤＮＡが用い
られ、遺伝子導入された形質転換体の検出法として用い
られる場合は、導入された外来遺伝子のＤＮＡが用いら
れ、また、遺伝病の場合は、塩基配列の−７−８が変異
したＤＮＡが用いられる。Here, the DNA detected in the present invention may be of any origin depending on the purpose of DNA detection. For example, in microbial testing, DNA derived from microorganisms is used, and when used as a method for detecting gene-introduced transformants, DNA of introduced foreign genes is used, and in the case of genetic diseases, DNA with a base sequence of -7 to 8 mutated is used.
本発明においては、公知の方法（生化学実験講座２、核
酸の化学■、４１〜５４ （１９７５） 、日本生化学
全編、東京化学同人発行）によってまず検体試料中の目
的とする微生物等からＤＮＡを抽出し、そのＤＮＡをＰ
ＣＲ法（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃ
ｔｉｏｎ法以下、５ａｉｋｉら、５ｃｉｅｎｃｅ、 ２
３０．１３５０ （１９８５））によって増幅する。こ
の増幅されたＤＮＡを含むＰＣＲ反応液を上層のミネラ
ルオイルを除いた後直接、キャピラリー電気泳動によっ
て分析する。In the present invention, DNA is first extracted from a target microorganism, etc. in a specimen sample by a known method (Biochemistry Experiment Course 2, Chemistry of Nucleic Acids, 41-54 (1975), Nippon Biochemistry, published by Tokyo Kagaku Doujin). Extract the DNA and P
CR method (Polymerase Chain Reac
tion method, 5aiki et al., 5science, 2
30.1350 (1985)). After removing the upper layer of mineral oil from the PCR reaction solution containing this amplified DNA, it is directly analyzed by capillary electrophoresis.
キャピラリー電気泳動は以下の方法によって行うが分析
条件及び操作方法等はこれに限定されない。Capillary electrophoresis is performed by the following method, but analysis conditions and operating methods are not limited thereto.
本発明に用いるキャピラリー電気泳動では、電気泳動用
緩衝液にゲル化していないポリマーを添加して電気泳動
を行うが、使用時にゲル化しないポリマーであればいか
なるものであっても使用することができる。例えば、ゲ
ル化温度が低いいわゆる低融点アガロース、デンプン等
が挙げられる。In capillary electrophoresis used in the present invention, electrophoresis is performed by adding a non-gelling polymer to the electrophoresis buffer, but any polymer that does not gel during use can be used. . Examples include so-called low melting point agarose, starch, etc., which have a low gelation temperature.
また、電気泳動用緩衝液には、ＳＤＳ （ドデシル硫
酸ナトリウム）、胆汁酸系等の界面活性剤を０．０１〜
０．５％添加することが好ましいが添加しなくてもよい
。In addition, the electrophoresis buffer contains surfactants such as SDS (sodium dodecyl sulfate) and bile acids at a concentration of 0.01 to
It is preferable to add 0.5%, but it may not be added.
また、電気泳動用緩衝液は例えば０．１Ｍのトリス（ヒ
ドロキシメチル）アミンメタン及びほう酸を緩衝剤とし
て含有するもの等が用いられるが、分離分析対象となる
試料に応じて種々の緩衝剤を用いることができる。The electrophoresis buffer used is one containing, for example, 0.1M tris(hydroxymethyl)aminemethane and boric acid as a buffer, but various buffers may be used depending on the sample to be separated and analyzed. I can do it.
キャピラリーの材質は、フユーズドシリカが好ましいが
これに限定されない。例えば、テフロンチューブ等であ
ってもよい。キャピラリーの内径は１０〜２００μｍが
好ましく、特に好ましくは５０〜１００μｍであるがこ
れに限定されない。The material of the capillary is preferably fused silica, but is not limited thereto. For example, it may be a Teflon tube or the like. The inner diameter of the capillary is preferably 10 to 200 μm, particularly preferably 50 to 100 μm, but is not limited thereto.
キャピラリーの長さは、通常３０〜５０ｃｍが用いられ
るがこれに限定されない。The length of the capillary is usually 30 to 50 cm, but is not limited thereto.
また、電源としては最大出力電圧３０ｋＶ程度のものが
好ましいがこれより小さいものであってもよく、これよ
り大きなものであってもよい。また、電流は直流が好ま
しいがパルス状に発生するものでもよくまた、これらに
限定されない。Further, the power source preferably has a maximum output voltage of about 30 kV, but it may be smaller or larger than this. Further, the current is preferably a direct current, but may be generated in a pulsed manner, and is not limited thereto.
また、検出器としては例えばＵＶ検出器あるいは蛍光検
出器が好ましいが電気化学検出器等であってもよく、ま
た、これらに限定されない。Further, as the detector, for example, a UV detector or a fluorescence detector is preferable, but an electrochemical detector or the like may also be used, and the detector is not limited to these.
また、記録計は保持時間、ピーク高、ピーク面積計算等
のデータ処理機能を持つものが好ましいがこれに限定さ
れない。Furthermore, the recorder preferably has data processing functions such as retention time, peak height, and peak area calculations, but is not limited thereto.
上記のポリマー等を含有する電気泳動用緩衝液を満たし
たキャピラリー内に端部から試料を導入し、キャピラリ
ーの両端をポリマー等を含有する電気泳動用緩衝液を入
れたそれぞれ別の電極槽に浸す。この二つの電極槽にそ
れぞれＰｔ電極を浸し両極に電圧を印加する。キャピラ
リー両端に電圧を印加することによってキャピラリー内
部のポリマー等を含む電気泳動用緩衝液に流れが生じ、
溶出された試料の成分を上記の検出器によって検出する
。検出器からの電気的な信号は記録計に伝達されそこで
処理される。A sample is introduced from the end into a capillary filled with an electrophoresis buffer containing the above polymer, etc., and both ends of the capillary are immersed in separate electrode baths filled with an electrophoresis buffer containing a polymer, etc. . A Pt electrode is immersed in each of these two electrode baths, and a voltage is applied to both electrodes. By applying a voltage to both ends of the capillary, a flow is generated in the electrophoresis buffer containing polymers inside the capillary.
The components of the eluted sample are detected by the above detector. Electrical signals from the detector are transmitted to a recorder and processed there.
［実施例］
以下の実施例により本発明のさらに詳細な説明を行うが
、本発明はこれらの実施例によって何等限定されるもの
ではない。[Examples] The present invention will be explained in more detail with reference to the following Examples, but the present invention is not limited to these Examples in any way.
を用いて適当な増菌培地に接種し、３７℃、好気的条件
下で終夜培養を行い、その培地１．５ｍｌから遠心操作
により菌体を回収した。１０ｍＭ）！Ｊス塩酸緩衝液（
ｐＨ７，５）で１回洗浄後、同緩衝液にリゾチームを１
ｍｇ／ｍｌとなるように溶かした液０゜５ｍｌで懸濁さ
せ、３７℃、ＩＯ分で溶菌させた。The cells were inoculated into an appropriate enrichment medium using the following method, cultured overnight at 37°C under aerobic conditions, and bacterial cells were collected from 1.5 ml of the medium by centrifugation. 10mM)! JS hydrochloric acid buffer (
After washing once with pH 7.5), add 1 lysozyme to the same buffer.
The cells were suspended in 0.5 ml of a solution containing mg/ml and lysed at 37° C. for IO minutes.
溶菌液に前記緩衝液で飽和させたフェノールを同容量加
え、よく撹はんした。遠心後、上層液を回収し、エタノ
ール沈澱処理を行って核酸成分を沈澱させ、その沈澱物
を前記緩衝液、１ｍｌに溶かして、これを試料とした。The same volume of phenol saturated with the above buffer was added to the lysate and stirred well. After centrifugation, the upper layer solution was collected and subjected to ethanol precipitation treatment to precipitate the nucleic acid component.The precipitate was dissolved in 1 ml of the above buffer solution and used as a sample.
２、ブライマーの台底
サルモネラ菌ＤＮＡを特異的に増幅するプライマーとし
て、下記の塩基配列をもつオリゴヌクレオチド（特願平
１−１８５６８３号）を化学合成した。2. An oligonucleotide (Japanese Patent Application No. 1-185683) having the following base sequence was chemically synthesized as a primer for specifically amplifying the Salmonella enterica DNA of the base of the primer.
（５°） ｄ−ＧＧＣＧＡＧＣＡＧＴＴＴＧＴＣＴＧＴ
Ｃ（３°）（５′） ｄ−ＧＴＴＴＣＧＣＣＴＧＧＣＴ
ＧＡＴＡＣＧ （３”〉化学合或は高滓ＤＮＡ台底装置
Ｎ５−１を用い、トリエステル法により行った。台底し
たオリゴヌクレオチドの精製はＣ１８逆相カラムを用い
て行った。(5°) d-GGCGAGCAGTTTGTCTGT
C (3°) (5') d-GTTTCGCCTGGCT
GATACG (3'') chemical synthesis was carried out by the triester method using a high-grade DNA platform apparatus N5-1. Purification of the oligonucleotides formed at the bottom was performed using a C18 reverse phase column.
３、 ＰＣＲ
前記試料液を３μｌ用いそれに滅菌蒸留水１６、０５μ
Ｒｓ １０ｘ反応用緩衝液３μＡＳｄＮＴＰ溶液４．８
μ１１ブライマーをそれぞれ１．５μｌそして耐熱性Ｄ
ＮＡポリメラーゼ０．１５μｌを加え３０μｌの反応液
を調製した。この反応液の入った容器にミネラルオイル
（ＳＩＧＭＡ社製）を５０μｌ加え反応液上に重層する
。各添加された液の内容を下記に示す。3. PCR Use 3 μl of the sample solution and add 16.05 μl of sterile distilled water to it.
Rs 10x reaction buffer 3μ ASdNTP solution 4.8
1.5 μl each of μ11 Brimer and heat resistant D
0.15 μl of NA polymerase was added to prepare a 30 μl reaction solution. Add 50 μl of mineral oil (manufactured by SIGMA) to the container containing this reaction solution and layer it on top of the reaction solution. The contents of each added liquid are shown below.
１０Ｘ反応用緩衝液； ５００ ｍＭ ＫＣＩ、
１００［１１Ｍ Ｔｒｉｓ−ＨＣＩ（ｐＨ８，３）、
１５ ｍＭ Ｍｇｃｌｚ、 ０．１％（ｗ／ｖ）ゼラチ
ン
ｄＮＴＰ溶液、 ｄＡＴＰ、 ｃｌｃＴＰ、 ｄＧＴ
Ｐ、 ｄＴＴＰを混合させたもので各最終濃度が１．２
５ ｍＭブライマー； 前述した化学合成精製品の各水
溶液（５００ｕｎｉｔ／ｍｌ＞
耐熱性ＤＮＡポリメラーゼ； Ｔａｑ ＤＮＡポリメ
ラーゼ（５ｕｎｉｔ／ｍｌ ； Ｐｅｒｋｉｎ Ｂｌ
ｍｅｒ Ｃｅｔｕｓ社製）反応条件は、次の通りである
。10X reaction buffer; 500 mM KCI,
100 [11M Tris-HCI (pH 8,3),
15 mM Mgclz, 0.1% (w/v) gelatin dNTP solution, dATP, clcTP, dGT
A mixture of P and dTTP, each with a final concentration of 1.2
5 mM Brimer; Each aqueous solution of the chemically synthesized purified product described above (500 units/ml> Thermostable DNA polymerase; Taq DNA polymerase (5 units/ml); Perkin Bl
mer Cetus) reaction conditions are as follows.
熱変性； ９４℃ １分
アニーリング； ３７℃ １分
重合反応； ６０℃ ■分
熱変性からアニーリングを経て重合反応に至る過程を１
サイクル（所要時間５．７分）とし、これを４２サイク
ル（所要時間約４時間）行った。これらの操作は、Ｐｅ
ｒｋｉｎ Ｈｏｍｅｒ Ｃｅｔｕｓ社製ＤＮＡ Ｔｈｅ
ｒｍａｌ Ｃｙｃｌｅｒに上記反応条件をプログラムす
ることにより行った。Thermal denaturation; 94℃ 1 minute annealing; 37℃ 1 minute polymerization reaction; 60℃ ■1 minute The process from thermal denaturation to annealing to polymerization reaction
cycle (required time: 5.7 minutes), and 42 cycles (required time: approximately 4 hours) were performed. These operations
rkin Homer Cetus DNA The
The reaction was carried out by programming the above reaction conditions into Rmal Cycler.
８０ｍ１の蒸留水に０．５ｇの低融点アガロースを加え
、よく撹はんした後加熱し溶解させ放冷後、これに１２
１ ｇのトリス（ヒドロキシメチル）アミノメタン、９
３ｍｇのエチレンジアミン四酢酸二ナトリウム、２０ｍ
ｇのドデシル硫酸ナトリウムおよび１００μｇのエチジ
ウムブロマイドを溶解させた。これにさらにほう酸を加
え、ｐＨを８．１に調型し、蒸留水を加えて正確に１０
０ｍ１にした。Add 0.5 g of low melting point agarose to 80 ml of distilled water, stir well, heat to dissolve, let cool, and add 12 g of low melting point agarose to 80 ml of distilled water.
1 g tris(hydroxymethyl)aminomethane, 9
3mg disodium ethylenediaminetetraacetate, 20m
g of sodium dodecyl sulfate and 100 μg of ethidium bromide were dissolved. Add boric acid to this, adjust the pH to 8.1, add distilled water and adjust the pH to 8.1.
I set it to 0m1.
第１Ｅｆｆｌに示したシステムを用いてキャピラリー電
気泳動を行った。すなわち、蛍光検出器（１）はＲＦ−
５４０型（島津製作所製、励起波長３００ｎｍ、検出波
長５９０ｎｍに設定）、高電圧電源（２〉はＨＥＲ３０
Ｐ Ｏ，１６−３ｌ型（検定プレシジョンデバイセズ
製）、記録計（３）はＣ’−Ｒ４Ａ型（島津製作所製）
、電極（４）はＰｔ線（０，５ｍｍφ−３０ｍｍ） 、
電極槽（５）は１．５ｍｌのサンプリングチューブを用
いた。Capillary electrophoresis was performed using the system shown in the first Effl. That is, the fluorescence detector (1) is RF-
540 type (manufactured by Shimadzu Corporation, excitation wavelength set to 300 nm, detection wavelength set to 590 nm), high voltage power supply (2> is HER30
P O, 16-3l type (manufactured by Precision Devices), recorder (3) is C'-R4A type (manufactured by Shimadzu Corporation)
, the electrode (4) is a Pt wire (0.5mmφ-30mm),
A 1.5 ml sampling tube was used for the electrode tank (5).
キャピラリー（６）は５ｃｉｅｎｔｉｆｉｃ Ｇｌａｓ
ｓＢｎｇｉｎｅｅｒｉｎｇ社のフユーズドシリ力キャピ
ラリーの内径７５μｍのものを使用した。キャビラＩＪ
−の全長は４５０ｍｍであり子種側から３００ｍｍの
所から２［［１ｍの幅で被覆を剥し、蛍光検出器に取り
付けた。このキャピラリー内には使用時に上記の低融点
アガロースを含有する電気泳動用緩衝液を満たし、両端
はそれぞれ低融点アガロースを含有する電気泳動用緩衝
液を入れた子種側電極槽及び極側電極槽に浸しておいた
。このとき二つの電極槽内の緩衝液の液面の高さが同じ
になるように調整しておいた。Capillary (6) is 5cientific Glass
A fused silica capillary manufactured by sBngineering with an inner diameter of 75 μm was used. Cabila IJ
The total length of - was 450 mm, and the coating was peeled off at a width of 2 m from 300 mm from the seed side and attached to a fluorescence detector. During use, this capillary is filled with an electrophoresis buffer containing the above-mentioned low-melting point agarose, and the progeny-side electrode tank and the pole-side electrode tank each contain an electrophoresis buffer containing low-melting-point agarose at both ends. I soaked it in At this time, the liquid levels of the buffer solutions in the two electrode tanks were adjusted to be the same height.
試料のキャピラリーへの導入はキャピラリーの＋極側の
端部を＋側電極槽から引き上げ試料溶液中に１０秒間浸
して行った。このとき試料の液面の高さは電極槽内の緩
衝液の液面より５０ｍｍ高くなるように調整して行った
。The sample was introduced into the capillary by lifting the + side end of the capillary from the + side electrode tank and immersing it in the sample solution for 10 seconds. At this time, the height of the liquid level of the sample was adjusted to be 50 mm higher than the liquid level of the buffer solution in the electrode tank.
試料をキャピラリー内に導入した後、キャピラリーの端
部を電極槽に戻しキャピラリーの両端に７、５ ｋＶの
直流電圧を印加した。電流値は１２〜１５μ八となり、
キャピラリー内には子種側から一極側に向かって緩衝液
の流れが生じ、ＤＮＡは分離され、溶出されて蛍光検出
器で検出された。ＰＣＲ反応によって増幅されたサルモ
ネラ菌ＤＮＡは期待される溶出時間にシャープなピーク
として検出された。この結果を第２図に示す。After introducing the sample into the capillary, the end of the capillary was returned to the electrode bath, and a DC voltage of 7.5 kV was applied to both ends of the capillary. The current value is 12~15μ8,
A buffer solution was generated in the capillary from the progeny side to the unipolar side, and the DNA was separated, eluted, and detected with a fluorescence detector. Salmonella bacteria DNA amplified by PCR reaction was detected as a sharp peak at the expected elution time. The results are shown in FIG.
［発明の効果コ
本発明の方法によれば、ＰＣＲ反応後の増幅されたＤＮ
Ａを含む溶液をゲルを作製することなしにキャピラリー
電気泳動を用いて簡便迅速かつ定量性再現性よく十分な
分離度で分析でき、しかもキャピラリー電気泳動に必要
なサンプル量が少なくて済むためＰＣＨの反応系を小さ
くすることができランニングコストを低く抑えることが
できる。[Effects of the Invention] According to the method of the present invention, the amplified DNA after PCR reaction
A-containing solutions can be easily and quickly analyzed using capillary electrophoresis without creating a gel, with good quantitative reproducibility and sufficient resolution.Moreover, the amount of sample required for capillary electrophoresis is small, making it easy to analyze PCH. The reaction system can be made smaller and running costs can be kept low.
第１図は、キャピラリー電気泳動のシステムを、第２図
は、増幅されたサルモネラＤＮＡの蛍光検出器による検
出を示す。
１・・・蛍光検出器
２・・・高電圧電源
３・・・記録計
４・・・電極
５・・・電極槽
６・・・キャピラリーFIG. 1 shows a system for capillary electrophoresis, and FIG. 2 shows detection of amplified Salmonella DNA by a fluorescence detector. 1... Fluorescence detector 2... High voltage power supply 3... Recorder 4... Electrode 5... Electrode tank 6... Capillary
Claims (1)
キャピラリー電気泳動法によって行うことを特徴とする
ＤＮＡの検出方法。(1) Detection of DNA amplified by gene amplification method,
A method for detecting DNA, characterized in that it is carried out by capillary electrophoresis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2040303A JP2722752B2 (en) | 1990-02-21 | 1990-02-21 | DNA detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2040303A JP2722752B2 (en) | 1990-02-21 | 1990-02-21 | DNA detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03243858A true JPH03243858A (en) | 1991-10-30 |
| JP2722752B2 JP2722752B2 (en) | 1998-03-09 |
Family
ID=12576852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2040303A Expired - Lifetime JP2722752B2 (en) | 1990-02-21 | 1990-02-21 | DNA detection method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2722752B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05107226A (en) * | 1990-05-18 | 1993-04-27 | Univ Northeastern | Pulse electric field capillary tube electrophoresis |
| JP2011141297A (en) * | 1999-07-26 | 2011-07-21 | Applied Biosystems Llc | Method and apparatus for reducing peak broadening accompanied by establishment of an electric field |
| JP2015114150A (en) * | 2013-12-10 | 2015-06-22 | 株式会社島津製作所 | Electrophoretic separation method |
-
1990
- 1990-02-21 JP JP2040303A patent/JP2722752B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| ANALITICAL CHEMISTRY=1987 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05107226A (en) * | 1990-05-18 | 1993-04-27 | Univ Northeastern | Pulse electric field capillary tube electrophoresis |
| JP2011141297A (en) * | 1999-07-26 | 2011-07-21 | Applied Biosystems Llc | Method and apparatus for reducing peak broadening accompanied by establishment of an electric field |
| JP2011174951A (en) * | 1999-07-26 | 2011-09-08 | Applied Biosystems Llc | Method for reducing peak broadening associated with establishment of electric field, and apparatus |
| JP2015114150A (en) * | 2013-12-10 | 2015-06-22 | 株式会社島津製作所 | Electrophoretic separation method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2722752B2 (en) | 1998-03-09 |
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