JPS63307362A - Simple detection of dna - Google Patents
Simple detection of dnaInfo
- Publication number
- JPS63307362A JPS63307362A JP14215787A JP14215787A JPS63307362A JP S63307362 A JPS63307362 A JP S63307362A JP 14215787 A JP14215787 A JP 14215787A JP 14215787 A JP14215787 A JP 14215787A JP S63307362 A JPS63307362 A JP S63307362A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- fluorescent dye
- compd
- detection
- spacer
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 3
- 150000001875 compounds Chemical class 0.000 claims description 8
- -1 spacer compound Chemical class 0.000 claims description 4
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 125000006850 spacer group Chemical group 0.000 abstract description 8
- 238000002372 labelling Methods 0.000 abstract description 6
- VSZWLDAGOXQHNB-UHFFFAOYSA-M 2-aminoethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCN VSZWLDAGOXQHNB-UHFFFAOYSA-M 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 150000003242 quaternary ammonium salts Chemical class 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 abstract 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- 230000021615 conjugation Effects 0.000 abstract 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 230000001268 conjugating effect Effects 0.000 abstract 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はＤＮＡの簡易検出法に関する。更に詳しくは、
標１１ＤＮ＾を用いるＤＮＡの簡易検出法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a simple method for detecting DNA. For more details,
This article relates to a simple method for detecting DNA using standard 11DN^.
〔従来の技術〕及び〔発明が解決しようとする問題点〕
分子生物学や遺伝子工学の分野で、目的とするＤＮＡ断
片あるいはそのＤＮＡを含んだ菌体を検出することは、
欠かすことが出来ない重要な手法である。[Prior art] and [Problems to be solved by the invention]
In the fields of molecular biology and genetic engineering, detecting a target DNA fragment or a bacterial cell containing that DNA is a
This is an important method that cannot be overlooked.
そのために、予め標識となるＤＮＡを種々の方法で標識
し、それをプローブ（釣り針）として、多種のＤＮＡが
混在しているものとハイブリッドを形成させ（ハイブリ
ダイゼーション）、そのプローブと相補するもの、すな
わち同種のＤＮＡを釣り上げる方法をとる。菌体の場合
は、同種のＤＮＡを含む菌体を釣り上げることになる。To do this, we pre-label DNA to be labeled using various methods, use it as a probe (fishhook), and form a hybrid with a mixture of various types of DNA (hybridization). In other words, a method is used to fish for DNA of the same species. In the case of bacterial cells, bacterial cells containing the same type of DNA are picked up.
ＤＮＡの標識法としては、放射性同位元素（Ｒ１）を用
いる方法が感度も良く多く使われているが、その安全性
や廃棄の問題から、設備上及び使用上の制限も多い。As a DNA labeling method, a method using a radioactive isotope (R1) is often used because of its good sensitivity, but there are many limitations regarding equipment and use due to safety and disposal issues.
そこで最近では、非放射性のａ識として、ＤＮＡを修飾
して抗原抗体反応によりある種の酵素を結合させ、発色
基質により検出する方法、あるいは数段の化学反応によ
り発色基質をつけ、それによる検出方法等が行われてい
るが、いずれの場合も工程が数段に及び煩雑さを伴う。Recently, as a non-radioactive detection method, methods have been developed in which DNA is modified and a certain type of enzyme is bound to it through an antigen-antibody reaction, and then detected using a chromogenic substrate. Alternatively, a chromogenic substrate is attached through several chemical reactions, and detection is performed using that method. Several methods have been used, but in each case, the process involves several steps and is complicated.
そこで本発明者は、上記問題点を解決するため鋭意検討
を行った結果、蛍光色素化合物を用いてＤＮＡを標識す
ることを見出し、本発明に至った。Therefore, the present inventor conducted intensive studies to solve the above problems, and as a result, discovered that DNA can be labeled using a fluorescent dye compound, leading to the present invention.
従って、本発明はＤＮＡの簡易検出法に係り、このＤＮ
Ａの簡易検出法は、標識ＤＮＡをプローブとして用い、
検出さるべきＤＮＡとの間にハイブリッドを形成させた
後に検出を行うＤＮＡの検出法において、標識ＤＮＡと
して、スペーサー化合物を介して蛍光色素化合物をイオ
ン結合させた［）ＮＡを用い、蛍光により検出を行うこ
とを特徴とする。Therefore, the present invention relates to a simple method for detecting DNA.
A simple detection method uses labeled DNA as a probe,
In a DNA detection method in which detection is performed after forming a hybrid with the DNA to be detected, [)NA to which a fluorescent dye compound is ionically bonded via a spacer compound is used as the labeled DNA, and detection is performed by fluorescence. It is characterized by doing.
蛍光色素化合物をＤＮＡの末端に直接結合させた場合、
得られた標９ＤＮＡの感度に懸念が残るので。When a fluorescent dye compound is directly attached to the end of DNA,
There remain concerns about the sensitivity of the obtained standard 9 DNA.
スペーサーを介してＤＮＡのリン酸基部分にイオン結合
で結合させ、それをプローブとして目的のＤＮＡを検出
する。It is bound to the phosphate group of DNA through a spacer through an ionic bond, and is used as a probe to detect the DNA of interest.
スペーサーとしては、４級アンモニウム塩などが用いら
れ１例えば一般式
で表わされるもの、具体的には２−アミノエチルトリメ
チルアンモニウムクロライド（ＡＥＴＡ）などが用いら
れる。As the spacer, a quaternary ammonium salt or the like is used, such as one represented by the general formula, specifically 2-aminoethyltrimethylammonium chloride (AETA).
蛍光色素化合物としては１次式
で表わされるフルオレセインイソチオシアネート（ＦＩ
ＴＣ）の様なアミノ基指向性のものが用いられる。As a fluorescent dye compound, fluorescein isothiocyanate (FI
An amino group-directed one such as TC) is used.
スペーサーと蛍光色素化合物とは、ｐｉ（９，０以上の
アルカリ側で、室温下、１時間位で容易に反応し、次式
の蛍光標識ＦＩＴＣ−ＡＥＴＡとなる。The spacer and the fluorescent dye compound easily react in about 1 hour at room temperature on the alkali side of pi (9.0 or more), resulting in the fluorescent label FITC-AETA of the following formula.
ＣＨ３
（式中、Ｆは前記式（２）におけるフルオレセイン部分
を表す）
この蛍光標識を、標識となるＤＮＡと室温で約半日間Ｐ
Ｈ約８．０付近に保持しつつ混合すると、 ＤＮＡのリ
ン酸基とイオン結合を起こし、標識ＤＮＡを生成する。CH3 (wherein F represents the fluorescein moiety in formula (2) above) This fluorescent label was incubated with DNA to be labeled for about half a day at room temperature.
When mixed while maintaining H around 8.0, ionic bonding occurs with the phosphate group of DNA, producing labeled DNA.
目的とするＤＮＡの検出は、通常のサザンハイプリダイ
ゼーションあるいはコロニーハイブリダイゼーションを
行った後、蛍光による検出、一般には蛍光顕微鏡で蛍光
を発しているものを選んで行われる。Detection of the target DNA is carried out by conventional Southern hybridization or colony hybridization, followed by fluorescence detection, generally by selecting those that emit fluorescence using a fluorescence microscope.
ＦＩＴＣの場合、励起波長（４９０ｎ＋ｍ）と蛍光波長
（５２０ｎｍ）が近いので、 ３９０ｎｎ＋の光をあて
てｗ４察する。In the case of FITC, the excitation wavelength (490n+m) and fluorescence wavelength (520nm) are close, so w4 is detected by applying 390nn+ light.
ＦＩＴＣ以外の蛍光色素化合物としては、次式で表わさ
れるテトラメチルローダミンイソチオシアネート（ＴＲ
ＩＴＣ）あるいは、次式で表わされるフルオレスカミン
、又は。−フタルアルデヒド
等が挙げられる。Examples of fluorescent dye compounds other than FITC include tetramethylrhodamine isothiocyanate (TR
ITC) or fluorescamine represented by the following formula, or. - Phthalaldehyde and the like.
蛍光色素化合物をスペーサーを介してイオン結合法でＤ
ＮＡに結合させ、’Ｒｒｆｔｏｕｈとして用いることに
より、目的のＤＮＡの検出の工程が簡略化され。A fluorescent dye compound is attached via a spacer using an ionic bonding method.
By binding to NA and using it as 'Rrftouh, the process of detecting the target DNA is simplified.
多数検体を簡便に検出することが出来る。Multiple samples can be detected easily.
大腸菌ｐＢＲ３２２由来の、プラスミドｐＥＨ５（９，
Ｉ Ｋｂ。Plasmid pEH5 (9,
I Kb.
Ｔｃ”）の塩基配列の分った部分について合成オリゴヌ
クレオチドによる変異を与えた。変異を与えられたプラ
スミドを選択するために、サンプルをＣａＣＱ、法によ
り大腸菌に形質転換し、プレート上にコロニーを形成さ
せた。A synthetic oligonucleotide was used to mutate the known part of the nucleotide sequence of Tc"). In order to select the mutated plasmid, the sample was transformed into E. coli by the CaCQ method, and colonies were plated. formed.
標ｌＤＮＡとして、変異を与えた合成オリゴヌクレオチ
ドに前記式（３）のＦＩＴＣ−ＡＥＴＡで標識した。As a reference DNA, a mutated synthetic oligonucleotide was labeled with FITC-AETA of the formula (3).
合成オリゴヌクレオチド中の、リン酸基８〜ｌＯ個当り
、ＦＩＴＣ１個となるように、ＦＩＴＣ−ＡＥＴＡを用
いた。FITC-AETA was used so that there was one FITC for every 8 to 10 phosphate groups in the synthetic oligonucleotide.
この標識ＤＮＡをプローブとして、コロニーハイブリダ
イゼーションを常法により行った後、蛍光顕微鏡で検出
し、光っているコロニーを選んだ。Using this labeled DNA as a probe, colony hybridization was performed in a conventional manner, and then detected using a fluorescence microscope, and glowing colonies were selected.
それらのコロニーからプラスミドを抽出し、塩基配列を
調べたところ変異が与えられていることが分った。When plasmids were extracted from these colonies and their base sequences were examined, they were found to have been mutated.
Claims (1)
ＮＡとの間にハイブリッドを形成させた後に検出を行う
ＤＮＡの検出法において、標識ＤＮＡとして、スペーサ
ー化合物を介して蛍光色素化合物をイオン結合させたＤ
ＮＡを用い、蛍光により検出を行うことを特徴とするＤ
ＮＡの簡易検出法。 ２、スペーサー化合物が４級アンモニウム塩化合物であ
る特許請求の範囲第１項記載のＤＮＡの簡易検出法。 ３、蛍光色素がアミノ基指向性試薬である特許請求の範
囲第１項記載のＤＮＡの簡易検出法。 ４、蛍光による検出が蛍光顕微鏡を用いて行われる特許
請求の範囲第１項記載のＤＮＡの簡易検出法。[Claims] 1. D to be detected using labeled DNA as a probe
In a DNA detection method in which detection is performed after forming a hybrid with NA, D to which a fluorescent dye compound is ionically bonded via a spacer compound is used as the labeled DNA.
D, characterized in that detection is performed by fluorescence using NA.
A simple method for detecting NA. 2. The simple DNA detection method according to claim 1, wherein the spacer compound is a quaternary ammonium salt compound. 3. The simple method for detecting DNA according to claim 1, wherein the fluorescent dye is an amino group-directed reagent. 4. The simple DNA detection method according to claim 1, wherein the fluorescence detection is performed using a fluorescence microscope.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62142157A JPH0827281B2 (en) | 1987-06-09 | 1987-06-09 | Simple DNA detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62142157A JPH0827281B2 (en) | 1987-06-09 | 1987-06-09 | Simple DNA detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63307362A true JPS63307362A (en) | 1988-12-15 |
| JPH0827281B2 JPH0827281B2 (en) | 1996-03-21 |
Family
ID=15308688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62142157A Expired - Lifetime JPH0827281B2 (en) | 1987-06-09 | 1987-06-09 | Simple DNA detection method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0827281B2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59151060A (en) * | 1983-01-24 | 1984-08-29 | ベクトン・ディッキンソン・アンド・カンパニ− | Chromogen tracer for assay |
| JPS61164160A (en) * | 1985-01-10 | 1986-07-24 | モレキユラー・ダイアグノステイツクス・インコーポレーテツド | Photochemical labelling method of nucleic acid for detectionby hybrid analysis |
-
1987
- 1987-06-09 JP JP62142157A patent/JPH0827281B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59151060A (en) * | 1983-01-24 | 1984-08-29 | ベクトン・ディッキンソン・アンド・カンパニ− | Chromogen tracer for assay |
| JPS61164160A (en) * | 1985-01-10 | 1986-07-24 | モレキユラー・ダイアグノステイツクス・インコーポレーテツド | Photochemical labelling method of nucleic acid for detectionby hybrid analysis |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0827281B2 (en) | 1996-03-21 |
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