JPH01152352A - Detection of mutagen - Google Patents
Detection of mutagenInfo
- Publication number
- JPH01152352A JPH01152352A JP62309741A JP30974187A JPH01152352A JP H01152352 A JPH01152352 A JP H01152352A JP 62309741 A JP62309741 A JP 62309741A JP 30974187 A JP30974187 A JP 30974187A JP H01152352 A JPH01152352 A JP H01152352A
- Authority
- JP
- Japan
- Prior art keywords
- mutagen
- dna
- density
- adsorbed
- gel
- 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
- 239000003471 mutagenic agent Substances 0.000 title claims abstract description 31
- 231100000707 mutagenic chemical Toxicity 0.000 title claims abstract description 30
- 230000003505 mutagenic effect Effects 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title claims abstract description 9
- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 9
- 238000002835 absorbance Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 16
- 230000001678 irradiating effect Effects 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 abstract description 11
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 abstract description 6
- 239000007984 Tris EDTA buffer Substances 0.000 abstract description 4
- 238000001962 electrophoresis Methods 0.000 abstract description 4
- 239000011543 agarose gel Substances 0.000 abstract description 3
- 239000000499 gel Substances 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 3
- 229920000936 Agarose Polymers 0.000 abstract description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 abstract description 2
- 238000011088 calibration curve Methods 0.000 abstract description 2
- -1 dexitrane Polymers 0.000 abstract description 2
- 229920002401 polyacrylamide Polymers 0.000 abstract description 2
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 241000385736 bacterium B Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000013120 recombinational repair Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、変異原物質の検出方法に関する。更に詳しく
は、変異原物質を迅速に検出し得る方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for detecting mutagens. More specifically, the present invention relates to a method capable of rapidly detecting mutagens.
現在用いられている変異原物質のスクリーニング方法と
しては１次のような方法が代表的なものとして挙げられ
る。The following primary method is representative of currently used screening methods for mutagens.
Ｒｅｃ−ａｓｓａｙ法：枯草菌を用いる方法で、変異原
物質により組換えあるいはＤＮＡ修復を行なうことので
きる菌Ｂ、 ｓｕｂ、 （Ｒｅｃ＋）とこのような機能
を行なうことのできない菌Ｂ、 ｓｕｂ、 （Ｒｅｃ−
）の２種を用い、変異原物質が存在するとＲｅｃ”は変
異を与えられるため増殖し、一方Ｒｅｃ−は死滅すると
いう原理に基いて変異原物質のスクリーニングが行われ
る。Rec-assay method: A method using Bacillus subtilis, in which a bacterium B, sub, (Rec+) that can perform recombination or DNA repair using a mutagen, and a bacterium B, sub, (Rec+) that cannot perform such functions. Rec-
) are used to screen for mutagens based on the principle that in the presence of a mutagen, Rec'' is mutated and proliferates, while Rec- is killed.
Ａｇｅｓ法：原理的にはＲｅｃ−ａｓｓａｙ法とほぼ同
じであるが、枯草菌ではなくサルモネラ菌を使用する点
で異なり、また株の特性としては、ヒスチジン要求性か
あるいは非要求性かで変異原物質のスクリーニングが行
われる。Ages method: In principle, it is almost the same as the Rec-assay method, but it differs in that Salmonella enterica is used instead of Bacillus subtilis, and the characteristics of the strain include whether it is a histidine-requiring or non-requiring mutagen. Screening will be carried out.
しかしながら、これらの方法を用いての変異原物質の検
出には、２〜３日間程度の期間とこれを行なうための熟
練とが必要となる。However, detection of mutagens using these methods requires a period of about 2 to 3 days and skill.
そこで、本発明者は、変異原物質の迅速な検出方法を求
めて種々の検討を行ない、変異原物質がＤＮＡに吸着し
て突然変異をひき起すという最も基本的な点に着目し、
これにゲル電気泳動法を適用した後紫外線照射するとい
う方法により、上記課題の効果的な解決を図った。Therefore, the present inventor conducted various studies in search of a rapid detection method for mutagens, and focused on the most basic point that mutagens adsorb to DNA and cause mutations.
We attempted to effectively solve the above problem by applying gel electrophoresis to this and then irradiating it with ultraviolet rays.
従って、本発明は変異原物質の検出方法に係り、変異原
物質の検出は、変異原物質をデオキシリボ核酸（ＤＮＡ
）に吸着させ、変異原物質吸着デオキシリボ核酸にゲル
電気泳動法を適用した後、紫外線を照射することにより
行われる。Therefore, the present invention relates to a method for detecting a mutagen.
), applying gel electrophoresis to the mutagen-adsorbed deoxyribonucleic acid, and then irradiating it with ultraviolet light.
本発明方法にあっては、まずニトロソグアニジン、ペン
ツピレン、エチルメタンスルホン酸、亜硝酸、ヒドロキ
シルアミンなどの変異原物質をＤＮＡに吸着させること
が行われる。この吸着は、 ＤＮＡ濃度約５〜ｌＯｐ
ｇ／ｍ Ｑ、変異原物質１度約１０〜２０ＩＩＩＭのそ
れぞれの溶液を、室温条件下で約３〜６０分間混合する
ことにより行われる。この場合の溶媒としては１例えば
ＴＥ緩衝液［１０＋ｍＭ トリス（ヒドロキシメチル）
アミノ、メタン、（２−アミノ−２−ヒドロキシメチル
−１，３−プロパンジオールと１ ｍＭ ＥＤＴＡジナ
トリウム塩との混合物のｐＨを８．０に調整したちの］
などが用いられ、両者の溶媒は同一であることが好まし
い。In the method of the present invention, a mutagen such as nitrosoguanidine, pentsupyrene, ethylmethanesulfonic acid, nitrous acid, or hydroxylamine is first adsorbed onto DNA. This adsorption occurs at a DNA concentration of approximately 5 to 1 Op.
g/m Q, mutagen once about 10-20 IIIM of each solution is mixed for about 3-60 minutes under room temperature conditions. In this case, the solvent is 1, for example, TE buffer [10+mM Tris(hydroxymethyl)
amino, methane, (after adjusting the pH of a mixture of 2-amino-2-hydroxymethyl-1,3-propanediol and 1 mM EDTA disodium salt to 8.0)
etc., and it is preferable that both solvents are the same.
このようにして変異原物質を吸着させたＤＮＡの溶液は
、ゲル電気泳動法にかけられる。ゲル電気泳動法は現在
、たん白質の解析（分子量測定、構造解析など）に用い
られているばかりではなく、ＤＮＡの切断、結合確認、
塩基配列の決定、分子量測定などに広く用いられており
、例えばＤＮＡの解析においては発がん性物質であるエ
チレンジプロミドを吸着させ、それにゲル電気泳動法を
適用した後、紫外線を照射して検出することも行われて
いる。The DNA solution to which the mutagen has been adsorbed in this manner is subjected to gel electrophoresis. Gel electrophoresis is currently used not only for protein analysis (molecular weight measurement, structural analysis, etc.), but also for DNA cleavage, binding confirmation,
It is widely used for determining base sequences and measuring molecular weight. For example, in DNA analysis, it adsorbs the carcinogenic substance ethylene dipromide, applies gel electrophoresis to it, and then irradiates it with ultraviolet light for detection. This is also being done.
本発明方法においては、これらのＤＮＡ解析に用いられ
ているゲル電気泳動法の手法がほぼその適用され、即ち
アガロース、デキストラン、ポリアクリルアミドまたは
それらの橋架は結合物などのゲルを濃度約０．３〜１％
で用い、電圧約５０〜１００Ｖ、時間約３０〜６０分間
の条件下で電気泳動が行われる。In the method of the present invention, most of the gel electrophoresis techniques used in these DNA analyzes are applied, that is, a gel containing agarose, dextran, polyacrylamide, or a conjugate thereof is prepared at a concentration of about 0.3. ~1%
Electrophoresis is performed under conditions of a voltage of about 50 to 100 V and a time of about 30 to 60 minutes.
電気泳動終了後、ＤＮＡ−変異原物質の吸着したバンド
を刃物で切り出し、透析チューブに入れ再度電圧をかけ
てアガロースゲルから分離し、分離された溶液に紫外線
、例えば波長２５４ｎｍの紫外線を照射すると発色する
ので、変異原物質を定性的に検出することができ、ある
いはその吸光度を測定することにより検量線から定量的
な検出も可能となる。After electrophoresis, the band with the DNA-mutagen adsorbed is cut out with a knife, placed in a dialysis tube and separated from the agarose gel by applying voltage again. When the separated solution is irradiated with ultraviolet light, for example, ultraviolet light with a wavelength of 254 nm, color develops. Therefore, the mutagen can be detected qualitatively, or quantitatively detected from the calibration curve by measuring its absorbance.
本発明方法によれば、変異原物質をＤＮＡに吸着させ、
それにゲル電気泳動法を適用した後紫外線照射するだけ
であるので、迅速に変異原物質の定性的あるいは定量的
な検出が可能となる。According to the method of the present invention, a mutagen is adsorbed to DNA,
Since it is only necessary to apply gel electrophoresis and then irradiate with ultraviolet rays, it is possible to rapidly qualitatively or quantitatively detect mutagens.
しかも、本発明方法は、変異原物質の迅速な検出を可能
とするばかりではなく、検出の再現性お′よび定量性を
向上させた点に特徴を有している。Furthermore, the method of the present invention is characterized in that it not only enables rapid detection of mutagens, but also improves the reproducibility and quantitative nature of detection.
即ち、再現性に関していえば、いずれも微生物を使用し
ている従来法と比較して確実性にすぐれていることは確
かであり、また定量性に関していえば、未吸着ＤＮＡと
の吸光度との差から容易に変異原物質の定量が可能であ
る。In other words, in terms of reproducibility, it is certain that both methods are more reliable than conventional methods that use microorganisms, and in terms of quantitativeness, the difference in absorbance from that of unadsorbed DNA is Mutagenic substances can be easily quantified from
次に、実施例により本発明を説明する。 Next, the present invention will be explained by examples.
実施例
濃度５μｇ／＋ａΩのＤＮＡのＴＥ緩衝液溶液および濃
度１０ｍＭのニトロソグアニジンのＴＥ緩衝液溶液を１
ｍΩ宛混合し、室温下に３０分間放置した。これを、０
．６％アガロースゲルを用いて、５０ｖで１時間電気泳
動した購、前記の如くにして取り出されたゲルから分離
された溶液に波長２５４ｎｍの紫外線ランプを照射する
と、そこにオレンジ色の発色がみられた。Example A solution of DNA in TE buffer with a concentration of 5 μg/+aΩ and a solution of nitrosoguanidine with a concentration of 10 mM in TE buffer
The mixture was mixed at mΩ and left at room temperature for 30 minutes. This is 0
．． After electrophoresis was performed at 50V for 1 hour using a 6% agarose gel, when the solution separated from the gel taken out as described above was irradiated with an ultraviolet lamp with a wavelength of 254 nm, an orange color was observed. Ta.
比較例１〜２
実施例において、ニトロソグアニジン溶液を用いずにＤ
ＮＡ溶液を用いた場合（比較例１）あるいはニトロソグ
アニジン溶液の代りにＩＩＩＱの１０％エタノール水溶
液を用いた場合（比較例２）には、いずれも紫外線照射
での発色がみられなかった。Comparative Examples 1-2 In Examples, D without using nitrosoguanidine solution
When an NA solution was used (Comparative Example 1) or when a 10% ethanol aqueous solution of IIIQ was used instead of the nitrosoguanidine solution (Comparative Example 2), no color development was observed upon irradiation with ultraviolet rays.
また、各濃度のＤＮＡにそれぞれｌｏｍＭのニトロソグ
アニジンを加えたもの［Ｉ］または加えなかったもの［
１１１について、ＤＮＡ濃度と波長２５４ｎｍの紫外線
の吸光度との関係を測定すると、第１図のグラフに示さ
れるような関係が得られ１両者はＤＮＡ濃度が同一であ
るので、吸光度の差が変異原物質の量を示すことになる
。In addition, each concentration of DNA was added with lomM of nitrosoguanidine [I] or was not added [I].
When measuring the relationship between the DNA concentration and the absorbance of ultraviolet light with a wavelength of 254 nm for 111, a relationship as shown in the graph of Figure 1 was obtained.1 Since the DNA concentration of both is the same, the difference in absorbance is considered to be a mutagen. It shows the amount of a substance.
第１図は、被測定物質の濃度と紫外線吸光度との関係を
示すグラフである。FIG. 1 is a graph showing the relationship between the concentration of a substance to be measured and ultraviolet absorbance.
Claims (1)
せ、変異原物質吸着デオキシリボ核酸にゲル電気泳動法
を適用した後、紫外線を照射することを特徴とする変異
原物質の検出方法。 ２、波長２５４ｎｍの紫外線を照射し、吸光度の値から
変異原物質を定量的に検出する特許請求の範囲第１項記
載の変異原物質の検出方法。 ３、波長２５４ｎｍの紫外線を照射し、発色の有無によ
り変異原物質を定性的に検出する特許請求の範囲第１項
記載の変異原物質の検出方法。[Claims] 1. A method of producing a mutagen, which is characterized by adsorbing a mutagen to deoxyribonucleic acid (DNA), applying gel electrophoresis to the mutagen-adsorbed deoxyribonucleic acid, and then irradiating it with ultraviolet rays. Detection method. 2. The method for detecting a mutagen according to claim 1, which comprises irradiating ultraviolet light with a wavelength of 254 nm and quantitatively detecting the mutagen from the absorbance value. 3. The method for detecting a mutagen according to claim 1, which comprises irradiating ultraviolet rays with a wavelength of 254 nm and qualitatively detecting the mutagen based on the presence or absence of color development.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62309741A JP2600225B2 (en) | 1987-12-09 | 1987-12-09 | Methods for detecting mutagens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62309741A JP2600225B2 (en) | 1987-12-09 | 1987-12-09 | Methods for detecting mutagens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01152352A true JPH01152352A (en) | 1989-06-14 |
| JP2600225B2 JP2600225B2 (en) | 1997-04-16 |
Family
ID=17996741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62309741A Expired - Lifetime JP2600225B2 (en) | 1987-12-09 | 1987-12-09 | Methods for detecting mutagens |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2600225B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01291145A (en) * | 1988-05-18 | 1989-11-22 | Dainippon Printing Co Ltd | Method and device for detecting ultraviolet absorber or phosphor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS584304A (en) * | 1981-06-29 | 1983-01-11 | Kanto Tokushu Seikou Kk | Holder for cutting-off operation |
-
1987
- 1987-12-09 JP JP62309741A patent/JP2600225B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS584304A (en) * | 1981-06-29 | 1983-01-11 | Kanto Tokushu Seikou Kk | Holder for cutting-off operation |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01291145A (en) * | 1988-05-18 | 1989-11-22 | Dainippon Printing Co Ltd | Method and device for detecting ultraviolet absorber or phosphor |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2600225B2 (en) | 1997-04-16 |
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