JP3793287B2 - Method for isolating plasmid DNA - Google Patents
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- JP3793287B2 JP3793287B2 JP22122196A JP22122196A JP3793287B2 JP 3793287 B2 JP3793287 B2 JP 3793287B2 JP 22122196 A JP22122196 A JP 22122196A JP 22122196 A JP22122196 A JP 22122196A JP 3793287 B2 JP3793287 B2 JP 3793287B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、遺伝子工学の分野において最も基本的な操作の一つである核酸からプラスミドＤＮＡを単離する方法に関する。
【０００２】
【従来の技術】
従来より、大腸菌プラスミドベクターに目的のＤＮＡを組み込んで培養し、目的のＤＮＡを大量に調製する方法は、遺伝子工学分野において最も頻繁に行われている操作の一つである。現在、大腸菌からのプラスミドベクターの回収は、溶菌操作の後ゲノムＤＮＡと蛋白質等を取り除いてＲＮＡとプラスミドＤＮＡの混合物を得ることによって行われ、その方法としては、アルカリ−ＳＤＳ法をはじめとしていくつかの方法が知られている。ＲＮＡとプラスミドＤＮＡの混合物からさらに精製プラスミドＤＮＡを得るためには、イオン交換樹脂を用いたカラム分離法、塩化セシウム密度勾配超遠心法などが一般的に用いられている。しかしながら、塩化セシウム密度勾配超遠心法では、大がかりな装置で高い回転数で長時間遠心処理を行うため、細心の注意を要し、煩雑であり、また、経済的でなかった。
【０００３】
【発明が解決しようとする課題】
本発明は、ＲＮＡとプラスミドＤＮＡを含む溶液から特別な装置を必要とせずに簡単な操作で精製プラスミドＤＮＡを得る方法を提供することを目的とする。
【０００４】
【課題を解決するための手段】
本発明は、特定濃度で特定ｐＨ範囲のリン酸塩緩衝溶液を作用させると、プラスミドＤＮＡはリン酸カルシウム系化合物粒子に吸着されるのに対し、ＲＮＡは吸着されないことを見出し、この知見に基づいて完成したものである。
すなわち、本発明によるプラスミドＤＮＡの単離方法は、０．２〜０．４Ｍ、ｐＨ７〜９のリン酸塩緩衝液中にＣａ／Ｐ比が１．０〜２．０のリン酸カルシウム系化合物粒子を懸濁させ、この懸濁液中に、溶菌操作の後に得られるＲＮＡとプラスミドＤＮＡを含む溶液を添加することにより該リン酸カルシウム系化合物粒子にプラスミドＤＮＡを吸着させ、このプラスミドＤＮＡを吸着したリン酸カルシウム系化合物粒子を分離し、０．２〜０．４Ｍ、ｐＨ６以下のリン酸塩緩衝液と接触させてプラスミドＤＮＡを脱着させることを特徴とする。
【０００５】
本発明はまた、プラスミドＤＮＡを液体クロマトグラフィーによって単離する方法に関し、この方法は、Ｃａ／Ｐ比が１．０〜２．０のリン酸カルシウム系化合物粒子をクロマトグラフィー用カラムに充填し、０．２〜０．４Ｍ、ｐＨ７〜９のリン酸塩緩衝液で平衡化した後、溶菌操作の後に得られるＲＮＡとプラスミドＤＮＡを含む溶液及び０．２〜０．４Ｍ、ｐＨ７〜９のリン酸塩緩衝液を順次通水し、該リン酸カルシウム系化合物粒子にプラスミドＤＮＡを吸着させた後、０．２〜０．４Ｍ、ｐＨ６以下のリン酸塩緩衝液を通水してプラスミドＤＮＡを溶離することを特徴とする。
【０００６】
【発明の実施の形態】
本発明に用いるリン酸カルシウム系化合物は、Ｃａ／Ｐ比が１．０〜２．０のものであれば、特に制限はなく、例えば、Ｃａ10（ＰＯ4)6 （ＯＨ)2、Ｃａ10（ＰＯ4)6 Ｆ2 、Ｃａ10（ＰＯ4)6 Ｃl2、Ｃａ3(ＰＯ4)2 、Ｃａ2 Ｐ2 Ｏ7 及びＣａ4 Ｏ（ＰＯ4)2 のうちから選ばれた１種又は２種以上を使用することができる。これらのうち安定性や吸着性能の点からＣａ／Ｐ比が１．５〜１．８のものが好ましく、Ｃａ／Ｐ比が１．６７のハイドロキシアパタイトを主成分とするものが最も好ましい。フッ素アパタイトを用いる場合、全リン酸カルシウム系化合物中のフッ素含有率が５重量％以下であるのが好ましい。フッ素含有率が５重量％を超えると、フッ素の溶出が起こり好ましくない。これらのリン酸カルシウム系化合物は、公知の方法で合成することができる。
【０００７】
本発明においては、リン酸カルシウム系化合物粒子は、その平均粒径が１μｍ〜１ｍｍの範囲にあり、かつＢＥＴ法による比表面積が１〜３００ｍ2 ／ｇ、好ましくは５〜３００ｍ2 ／ｇの範囲内にあるものが好ましい。平均粒径が１μｍより小さいと、取扱いが困難であり、比表面積が１ｍ2 ／ｇより小さいと、低比表面積のため充分な吸着性能が得られない。一方、平均粒径が１ｍｍより大きく、比表面積が３００ｍ2 ／ｇより大きいと、それぞれ充填密度の低下、不均一な凝集体の発生などの理由から性能が劣化する。
上記のようなリン酸カルシウム系化合物は、種々のカルシウム化合物及びリン酸化合物を出発原料として合成し、造粒乾燥した後分級することにより製造することができる。上記の条件に適合した粒子を得るには、多孔質顆粒とすることが必要となることもあるが、多孔質顆粒は、常法で製造することができる。
【０００８】
本発明の方法においては、上記のようなリン酸カルシウム系化合物粒子に核酸成分中のプラスミドＤＮＡだけを吸着させるために、０．２〜０．４Ｍ、ｐＨ７〜９のリン酸塩緩衝液中にＣａ／Ｐ比が１．０〜２．０のリン酸カルシウム系化合物粒子を懸濁させ、この懸濁液中に、溶菌操作の後に得られるＲＮＡとプラスミドＤＮＡを含む溶液を添加する。溶菌操作は、アルカリ−ＳＤＳ法など、任意の公知の方法で行うことができる。溶菌操作により大腸菌中のゲノムＤＮＡや菌体の構成成分としての蛋白質や脂質を除去し、残りのプラスミドＤＮＡとＲＮＡを主として含む溶液を用いる。
リン酸塩緩衝液の濃度が０．２Ｍ未満であると、試料中のＲＮＡがプラスミドと共にリン酸カルシウム系化合物粒子に吸着される。また、０．４Ｍを超えると、リン酸塩が析出する恐れがある。。リン酸塩緩衝液が０．３〜０．４ＭでｐＨ７〜８である場合に、リン酸カルシウム系化合物粒子はプラスミドＤＮＡだけを吸着する能力が高く、好結果が得られる。
【０００９】
また、リン酸塩緩衝液のｐＨが７〜９の範囲にあるとき、プラスミドＤＮＡだけがリン酸カルシウム系化合物粒子に吸着され、ｐＨ７未満、特にｐＨ６以下のリン酸塩緩衝液を作用させると、リン酸カルシウム系化合物粒子に吸着されていたプラスミドＤＮＡが脱着される。これにより、プラスミドＤＮＡを核酸成分から容易に単離することができる。
したがって、上記のようなリン酸カルシウム系化合物粒子をクロマトグラフィー用カラムに充填しておき、液体クロマトグラフィーによってプラスミドＤＮＡを核酸成分から容易に単離することもできる。
【００１０】
【実施例】
次に、実施例に基づいて本発明をさらに詳細に説明するが、本発明はこれによって制限されるものではない。
【００１１】
実施例１〜３
（１）試料溶液の調製
アンピシリン耐性遺伝子を持った pBulescriptSK（＋）ベクターをＪＭ１０９コンピテントセルに形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ−寒天培地上で３７℃で一昼夜培養して単一コロニー株を得た。これを５０μｇ／ｍｌのアンピシリンを含むＬＢ培養液中で３７℃で一昼夜振盪培養して菌体を得た。この菌体を遠心分離したのち、アルカリ−ＳＤＳ法により核酸濃度２００μｇ／ｍｌのプラスミド−ＲＮＡ混合溶液（以下、試料溶液と称する。）を得た。
（２）吸着工程
平均粒径２０μｍ、比表面積５０ｍ2 ／ｇのハイドロキシアパタイト粒子２０ｍｇを表１に示すｐＨ及び濃度のリン酸ナトリウム緩衝液７０μｌ中に懸濁させ、試料溶液を１５μｌ添加し、室温で５分間振盪した。その後、懸濁液の上清を回収し、０．８％アガロースゲル電気泳動を行い、臭化エチジウム染色により核酸成分の分析（未吸着物質の確認）を行い、結果を表１に示す。
（３）脱着工程
上記懸濁液から分離したハイドロキシアパタイト粒子に、濃度が４００ｍＭでｐＨが６のリン酸ナトリウム緩衝液を加え、室温で５分間振盪し、上清を回収し、０．８％アガロースゲル電気泳動を行い、臭化エチジウム染色により核酸成分の分析（ハイドロキシアパタイト粒子から脱着された物質の確認）を行い、結果を表１に示す。
【００１２】
比較例１
平均粒径２０μｍ、比表面積５０ｍ2 ／ｇのハイドロキシアパタイト粒子２０ｍｇをｐＨ６の４００ｍＭリン酸ナトリウム緩衝液７０μｌ中に懸濁させ、実施例１（１）で調製した試料溶液を１５μｌ添加し、室温で５分間振盪した。その後、懸濁液の上清を回収し、０．８％アガロースゲル電気泳動を行い、臭化エチジウム染色により核酸成分の分析を行ったところ、プラスミドＤＮＡ及びＲＮＡの両方が上清に含まれており、ハイドロキシアパタイト粒子に吸着されなかったことが分かった。結果を表１に示す。
【００１３】
実施例４〜６
吸着工程において、表１に示す濃度及びｐＨのリン酸ナトリウム緩衝液を用い、脱着工程ではｐＨ６の３００ｍＭリン酸ナトリウム緩衝液を用いた以外は、実施例１と同様に操作し、結果を表１に示す。
【００１４】
比較例２
ｐＨ６の３００ｍＭリン酸ナトリウム緩衝液を用いた以外は、比較例１と同様に操作したところ、表１に示したように、プラスミドＤＮＡ及びＲＮＡもハイドロキシアパタイト粒子に吸着されなかった。
【００１５】
実施例７〜９
吸着工程において、表１に示す濃度及びｐＨのリン酸ナトリウム緩衝液を用い、脱着工程ではｐＨ６の２００ｍＭリン酸ナトリウム緩衝液を用いた以外は、実施例１と同様に操作し、結果を表１に示す。
【００１６】
比較例３
ｐＨ６の２００ｍＭリン酸ナトリウム緩衝液を用いた以外は、比較例１と同様に操作したところ、表１に示したように、プラスミドＤＮＡ及びＲＮＡもハイドロキシアパタイト粒子に吸着されなかった。
【００１７】
【表１】
表１に示した結果から明らかなとおり、０．２〜０．４Ｍ、ｐＨ７〜９のリン酸ナトリウム緩衝液を用いて高純度で精製プラスミドＤＮＡを得ることができたが、０．３〜０．４Ｍ、ｐＨ７〜８のリン酸ナトリウム緩衝液を用いた場合に、特に純度の高い精製プラスミドＤＮＡが得られた。
【００１９】
実施例１０
平均粒径２０μｍ、比表面積５０ｍ2 ／ｇのハイドロキシアパタイト粒子をクロマトグラフィー用カラムに充填し、３００ｍＭ、ｐＨ８のリン酸ナトリウム緩衝液でカラム内を洗い、実施例１（１）で調製した試料溶液を入れ、３００ｍＭ、ｐＨ８のリン酸ナトリウム緩衝液をカラム容積の３倍量通水した後、２００ｍＭ、ｐＨ６のリン酸ナトリウム緩衝液をカラム容積の２倍量通水し、これを溶離液とした。溶離液を回収し、０．８％アガロースゲル電気泳動を行い、臭化エチジウム染色により核酸成分の分析を行ったところ、プラスミドＤＮＡの存在を確認することができた。
【００２０】
【発明の効果】
本発明の方法によれば、溶菌操作によって得られる、ＲＮＡとプラスミドＤＮＡを含む溶液から特別な装置を必要とせずに簡単な操作で精製プラスミドＤＮＡを効率よく得ることができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for isolating plasmid DNA from nucleic acid, which is one of the most basic operations in the field of genetic engineering.
[0002]
[Prior art]
Conventionally, a method for preparing a large amount of target DNA by culturing by inserting the target DNA into an E. coli plasmid vector is one of the most frequently performed operations in the field of genetic engineering. Currently, recovery of plasmid vectors from E. coli is performed by removing genomic DNA and proteins after lysis and obtaining a mixture of RNA and plasmid DNA, including several alkali-SDS methods. The method is known. In order to obtain further purified plasmid DNA from a mixture of RNA and plasmid DNA, a column separation method using an ion exchange resin, a cesium chloride density gradient ultracentrifugation method and the like are generally used. However, in the cesium chloride density gradient ultracentrifugation method, the centrifugal treatment is performed for a long time at a high rotation speed with a large-scale apparatus, so it requires careful attention, is complicated, and is not economical.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for obtaining purified plasmid DNA from a solution containing RNA and plasmid DNA by a simple operation without requiring a special apparatus.
[0004]
[Means for Solving the Problems]
The present invention has been found based on the finding that when a phosphate buffer solution having a specific pH range is applied at a specific concentration, plasmid DNA is adsorbed to calcium phosphate compound particles, but RNA is not adsorbed. It is a thing.
That is, in the method for isolating plasmid DNA according to the present invention, calcium phosphate compound particles having a Ca / P ratio of 1.0 to 2.0 are contained in a phosphate buffer solution of 0.2 to 0.4 M and pH 7 to 9. The calcium phosphate compound in which the plasmid DNA is adsorbed by adsorbing the plasmid DNA to the calcium phosphate compound particles by adding a solution containing RNA and plasmid DNA obtained after the lysis operation to the suspension The particles are separated and contacted with a phosphate buffer having a pH of 0.2 to 0.4 M and pH 6 or less to desorb plasmid DNA.
[0005]
The present invention also relates to a method for isolating plasmid DNA by liquid chromatography, in which the calcium phosphate compound particles having a Ca / P ratio of 1.0 to 2.0 are packed in a chromatography column, and A solution containing RNA and plasmid DNA obtained after lysis operation after equilibration with a phosphate buffer of 2 to 0.4 M and pH 7 to 9 and a phosphate of 0.2 to 0.4 M and pH 7 to 9 Passing a buffer solution sequentially, adsorbing plasmid DNA to the calcium phosphate compound particles, and then passing a phosphate buffer solution of 0.2 to 0.4 M, pH 6 or less to elute the plasmid DNA. Features.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The calcium phosphate compound used in the present invention is not particularly limited as long as the Ca / P ratio is 1.0 to 2.0. For example, Ca 10 (PO 4 ) 6 (OH) 2 , Ca 10 (PO 4) 6 F 2, Ca 10 (PO 4) 6 Cl 2, Ca 3 (PO 4) 2, Ca 2 P 2 O 7 and Ca 4 O (PO 4) 1 or 2 or selected from among 2 The above can be used. Of these, those having a Ca / P ratio of 1.5 to 1.8 are preferred from the viewpoint of stability and adsorption performance, and those having hydroxyapatite as the main component having a Ca / P ratio of 1.67 are most preferred. When fluorapatite is used, the fluorine content in the total calcium phosphate compound is preferably 5% by weight or less. If the fluorine content exceeds 5% by weight, elution of fluorine occurs, which is not preferable. These calcium phosphate compounds can be synthesized by a known method.
[0007]
In the present invention, the calcium phosphate compound particles have an average particle diameter in the range of 1 μm to 1 mm and a specific surface area by the BET method of 1 to 300 m 2 / g, preferably in the range of 5 to 300 m 2 / g. Some are preferred. When the average particle size is less than 1 μm, handling is difficult, and when the specific surface area is less than 1 m 2 / g, sufficient adsorption performance cannot be obtained due to the low specific surface area. On the other hand, when the average particle size is larger than 1 mm and the specific surface area is larger than 300 m 2 / g, the performance deteriorates due to a decrease in packing density and generation of non-uniform aggregates.
The calcium phosphate compound as described above can be produced by synthesizing various calcium compounds and phosphate compounds as starting materials, granulating and drying and then classifying. In order to obtain particles that meet the above conditions, it may be necessary to form porous granules, but porous granules can be produced by conventional methods.
[0008]
In the method of the present invention, in order to adsorb only the plasmid DNA in the nucleic acid component to the calcium phosphate-based compound particles as described above, Ca / 0.2 in a phosphate buffer solution of 0.2 to 0.4 M, pH 7 to 9. Calcium phosphate compound particles having a P ratio of 1.0 to 2.0 are suspended, and a solution containing RNA and plasmid DNA obtained after lysis is added to the suspension. The lysis operation can be performed by any known method such as alkali-SDS method. A solution containing mainly the remaining plasmid DNA and RNA is used by removing proteins and lipids as components of the genomic DNA and bacterial cells in E. coli by lysis.
When the concentration of the phosphate buffer is less than 0.2 M, RNA in the sample is adsorbed to the calcium phosphate compound particles together with the plasmid. Moreover, when it exceeds 0.4M, there exists a possibility that a phosphate may precipitate. . When the phosphate buffer is 0.3 to 0.4 M and pH is 7 to 8, the calcium phosphate compound particles have a high ability to adsorb only plasmid DNA, and good results are obtained.
[0009]
Further, when the pH of the phosphate buffer is in the range of 7 to 9, only the plasmid DNA is adsorbed on the calcium phosphate compound particles, and when a phosphate buffer having a pH of less than 7, particularly pH 6 or less is allowed to act, The plasmid DNA adsorbed on the compound particles is desorbed. Thereby, plasmid DNA can be easily isolated from a nucleic acid component.
Accordingly, the calcium phosphate compound particles as described above are packed in a chromatography column, and the plasmid DNA can be easily isolated from the nucleic acid component by liquid chromatography.
[0010]
【Example】
Next, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.
[0011]
Examples 1-3
(1) Preparation of sample solution A pBulescriptSK (+) vector having an ampicillin resistance gene was transformed into JM109 competent cells and cultured on an LB-agar medium containing 50 μg / ml ampicillin overnight at 37 ° C. A colony strain was obtained. This was shaken and cultured overnight at 37 ° C. in an LB medium containing 50 μg / ml ampicillin to obtain bacterial cells. After centrifuging the cells, a plasmid-RNA mixed solution (hereinafter referred to as a sample solution) having a nucleic acid concentration of 200 μg / ml was obtained by alkali-SDS method.
(2) Adsorption process 20 mg of hydroxyapatite particles having an average particle size of 20 μm and a specific surface area of 50 m 2 / g are suspended in 70 μl of sodium phosphate buffer having the pH and concentration shown in Table 1, 15 μl of sample solution is added, and room temperature is added. And shaken for 5 minutes. Thereafter, the supernatant of the suspension was collected, subjected to 0.8% agarose gel electrophoresis, analyzed for nucleic acid components (confirmation of unadsorbed substances) by ethidium bromide staining, and the results are shown in Table 1.
(3) Desorption step To the hydroxyapatite particles separated from the suspension, a sodium phosphate buffer solution having a concentration of 400 mM and a pH of 6 is added, shaken at room temperature for 5 minutes, the supernatant is recovered, and 0.8% Agarose gel electrophoresis was performed, nucleic acid components were analyzed by ethidium bromide staining (confirmation of substances desorbed from hydroxyapatite particles), and the results are shown in Table 1.
[0012]
Comparative Example 1
20 mg of hydroxyapatite particles having an average particle size of 20 μm and a specific surface area of 50 m 2 / g are suspended in 70 μl of a 400 mM sodium phosphate buffer at pH 6, and 15 μl of the sample solution prepared in Example 1 (1) is added at room temperature. Shake for 5 minutes. Thereafter, the supernatant of the suspension was recovered, subjected to 0.8% agarose gel electrophoresis, and nucleic acid components were analyzed by ethidium bromide staining. As a result, both plasmid DNA and RNA were contained in the supernatant. It was found that it was not adsorbed on the hydroxyapatite particles. The results are shown in Table 1.
[0013]
Examples 4-6
The same procedure as in Example 1 was performed except that a sodium phosphate buffer solution having the concentration and pH shown in Table 1 was used in the adsorption step, and a 300 mM sodium phosphate buffer solution having a pH of 6 was used in the desorption step. Shown in
[0014]
Comparative Example 2
As shown in Table 1, plasmid DNA and RNA were not adsorbed to the hydroxyapatite particles, except that a 300 mM sodium phosphate buffer at pH 6 was used.
[0015]
Examples 7-9
The same procedure as in Example 1 was carried out except that a sodium phosphate buffer solution having the concentration and pH shown in Table 1 was used in the adsorption step, and a 200 mM sodium phosphate buffer solution having a pH of 6 was used in the desorption step. Shown in
[0016]
Comparative Example 3
As shown in Table 1, plasmid DNA and RNA were not adsorbed to the hydroxyapatite particles except that a 200 mM sodium phosphate buffer at pH 6 was used.
[0017]
[Table 1]
As is clear from the results shown in Table 1, purified plasmid DNA could be obtained with high purity using a sodium phosphate buffer solution of 0.2 to 0.4 M and pH 7 to 9, but 0.3 to 0 When a 4M sodium phosphate buffer of pH 7-8 was used, purified plasmid DNA with particularly high purity was obtained.
[0019]
Example 10
A sample solution prepared in Example 1 (1) was prepared by packing hydroxyapatite particles having an average particle diameter of 20 μm and a specific surface area of 50 m 2 / g into a chromatography column, washing the column with a 300 mM sodium phosphate buffer at pH 8. After passing 300 mM of sodium phosphate buffer of pH 8 through the column volume 3 times, 200 mM of pH 6 sodium phosphate buffer was passed through the column volume of 2 times, and this was used as the eluent. . The eluate was collected, subjected to 0.8% agarose gel electrophoresis, and nucleic acid components were analyzed by ethidium bromide staining. As a result, the presence of plasmid DNA could be confirmed.
[0020]
【The invention's effect】
According to the method of the present invention, purified plasmid DNA can be efficiently obtained from a solution containing RNA and plasmid DNA obtained by a lysis operation by a simple operation without requiring a special apparatus.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22122196A JP3793287B2 (en) | 1996-08-22 | 1996-08-22 | Method for isolating plasmid DNA |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22122196A JP3793287B2 (en) | 1996-08-22 | 1996-08-22 | Method for isolating plasmid DNA |
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| JPH1057056A JPH1057056A (en) | 1998-03-03 |
| JP3793287B2 true JP3793287B2 (en) | 2006-07-05 |
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| JP22122196A Expired - Fee Related JP3793287B2 (en) | 1996-08-22 | 1996-08-22 | Method for isolating plasmid DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB9425138D0 (en) | 1994-12-12 | 1995-02-08 | Dynal As | Isolation of nucleic acid |
| KR20010032806A (en) * | 1997-12-06 | 2001-04-25 | 매튜 존 베이커 | Isolation of nucleic acids |
| KR100478138B1 (en) * | 2002-10-28 | 2005-03-21 | 주식회사 코젠바이오텍 | Dna contained composition for keeping and method for storing dna |
| WO2004100336A1 (en) * | 2003-05-02 | 2004-11-18 | Abb Inc. | Intelligence automatic bypass for a motor control device fault |
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