JPS62236486A - Method for separating small-sized cyclic double-stranded satellite dna in procaryotic cell particle - Google Patents
Method for separating small-sized cyclic double-stranded satellite dna in procaryotic cell particleInfo
- Publication number
- JPS62236486A JPS62236486A JP61077853A JP7785386A JPS62236486A JP S62236486 A JPS62236486 A JP S62236486A JP 61077853 A JP61077853 A JP 61077853A JP 7785386 A JP7785386 A JP 7785386A JP S62236486 A JPS62236486 A JP S62236486A
- Authority
- JP
- Japan
- Prior art keywords
- stranded
- prokaryotic
- cell
- filter
- circular double
- 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.)
- Pending
Links
- 239000002245 particle Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 19
- 108020004487 Satellite DNA Proteins 0.000 title claims abstract description 12
- 125000004122 cyclic group Chemical group 0.000 title abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 239000000725 suspension Substances 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims abstract description 6
- 150000003839 salts Chemical class 0.000 claims abstract description 6
- 102000053602 DNA Human genes 0.000 claims abstract description 5
- 239000013612 plasmid Substances 0.000 claims abstract description 5
- 239000000706 filtrate Substances 0.000 claims abstract description 4
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 4
- 230000003362 replicative effect Effects 0.000 claims abstract description 3
- 241000700605 Viruses Species 0.000 claims abstract 2
- 102000004169 proteins and genes Human genes 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 16
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 5
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 5
- 210000002421 cell wall Anatomy 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 3
- 210000002706 plastid Anatomy 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 2
- 230000009089 cytolysis Effects 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 230000002934 lysing effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 17
- 239000007788 liquid Substances 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 2
- 210000003763 chloroplast Anatomy 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 6
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 6
- 229960005542 ethidium bromide Drugs 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- SMWALVPHKGXESK-BMIPBODUSA-N 4-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-3-[[(2s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-2,4-diamino-4-oxobutanoyl]amino]-3-(4-sulfooxyphenyl)propanoyl]amino]-3-(4-sulfooxyphenyl)propanoyl]amino]acetyl]amino]-3-(1h-indol-3-yl)propanoyl]amino]-4- Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)NC(CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)NC(=O)[C@@H](N)CC(N)=O)C1=CC=C(OS(O)(=O)=O)C=C1 SMWALVPHKGXESK-BMIPBODUSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-O Htris Chemical compound OCC([NH3+])(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-O 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 108010056039 cionin Proteins 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 229920000131 polyvinylidene Polymers 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 210000000779 thoracic wall Anatomy 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using 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、原核細胞並びに、真核細胞から取得したミト
コンドリアや集緑体等の、原核細胞型粒子から、プラス
ミドや、ウィルスのリプリケイティブフォーム等の、小
型環状二本鎖ＤＮＡを分取する方法に関する。Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to the production of plasmids and viral replicative particles from prokaryotic cell-type particles such as mitochondria and plastids obtained from prokaryotic cells and eukaryotic cells. The present invention relates to a method for fractionating small circular double-stranded DNA such as foam.
（従来の技術）
従来、原核細胞型粒子内の、小型環状二本鎖サテライト
ＤＮＡを分取する方法は、遺伝子操作に不可欠な技術で
あり、組換えＤＮＡに用いるベクターを取得する手段と
して、あるいは小型環状二本鎖サテライトＤＮＡそのも
のをωＩ究する場合の試料の取得の手段として多用され
ており、その方法も多かった、しかしそれら従来の方法
は、いずれも遠心分１１１を用いていた、ここに−例と
して最も多く使用される方法の一つで、Ｔ、 Ｈａｎｉ
ａｔｉＳ等によッテ、米国のＣｏ１ｄ ５ｐｒｉｎｏ
ｌｌａｒｂｏｒ社から１９８２年に出版されたｒＨｏｌ
ｅｃｕｌａｒ ＣｌｏｎｉｎｇＪに記載された方法を示
すが、この方法は、特に大腸菌からプラスミドを精製す
る方法として記載されていて、
１〉大腸の培養を４０００ｘｇで１０分間４℃で遠沈し
、上清を捨てる工程、
２）緩衝液、この場合は０． Ｉ ＭＮａＣｅ、 １０
ａ＋ＨＴｒｉｓ ＰＨ７、８，１１ＨＥＤＴＡを注ぎ
、菌体を再懸濁し、再度４０００Ｘ９で１０分間４℃で
遠沈 ゛し、上清を拾でることによって洗浄する工程、
３）界面活性剤ＴｒｉｔｏｎＸ−１００を含む緩衝液に
再懸濁し、細胞壁分解酵素１ｙｓｏｚｙｎ＋ｅの溶液を
加える工程、
４）」−２３）で得られた液を加熱し、溶菌させる工程
、
５）得られた溶菌液を急冷する工程、
６）冷えた溶菌液を、超遠心分離機で、２５０００ｒｐ
ｍで、３０分間遠沈し、上清を取得する工程、７）６）
で得た上清に塩化セシウムを加え、密度を１．５５１７
／ａｔｅに調整し、エチジウムブロマイドを加える工程
、
８）超遠心分離機で４５０００ ｒｐｍで３６時間２０
℃で遠心回転する工程、
９）エチジウムブロマイドで染色された分画を取得する
工程、および
１０）ブタノールでエチジウムブロマイドを抽出する工
程、
から成る方法であり、他の方法も遠心分離機を多用する
点で類似しているがその様な例は、口。(Prior art) Conventionally, the method of separating small circular double-stranded satellite DNA in prokaryotic particles is an indispensable technique for genetic manipulation, and has been used as a means to obtain vectors for use in recombinant DNA, or It is often used as a means of obtaining samples when investigating small circular double-stranded satellite DNA itself for ωI, and there are many methods for doing so. However, all of these conventional methods used centrifugation. - One of the most used methods as an example, T. Hani
atiS et al., Co1d 5prino in the United States
rHol published in 1982 by llarbor
The method described in Ecular CloningJ is shown, and this method is specifically described as a method for purifying plasmids from E. coli. 1> Centrifuge the colon culture at 4000 x g for 10 minutes at 4°C, and discard the supernatant. Step 2) Buffer, in this case 0. I MNaCe, 10
Pour a+HTris PH7,8,11HEDTA, resuspend the bacterial cells, centrifuge again at 4000X9 for 10 minutes at 4°C, and wash by collecting the supernatant,
3) resuspending in a buffer containing surfactant Triton 6) The cooled lysate was heated at 25,000 rpm using an ultracentrifuge.
step of centrifuging at m for 30 minutes and obtaining supernatant, 7) 6)
Add cesium chloride to the supernatant obtained in , and adjust the density to 1.5517.
/ate and adding ethidium bromide, 8) In an ultracentrifuge at 45,000 rpm for 36 hours 20
This method consists of the following steps: 9) obtaining a fraction stained with ethidium bromide; and 10) extracting ethidium bromide with butanol; other methods also make extensive use of centrifuges. An example of this, but similar in some respects, is the mouth.
Ｈ，ＧＩＯＶｅｉｒによって編集された「ＤＨＡ Ｃ１
ｏｎｉｎａ Ｊ（（ＲＬ Ｐｒｅｓｓ ）やＢ、 Ｐｅ
ｒｂａｌによって著わされた「＾Ｐｒａｃｔｉｃａｌ
Ｇｕｉｄｅ ｔｏ Ｍｏ１ｅｃｕｌａｒ ＣＩｏｎｉｎ
ｇＪ（Ｗｉｌｅｙ ＩｎｔＯｒＳＣｉＯｎＣＱ）や、Ｒ
，Ｆ、 ｓｃｈ＋ｃｔｒとＰ、 Ｃ，Ｗｅｎｓｉｎｋに
よって著わされた［ｐｒａｃｔｔｃａｔＭｅｔｈｏｄｓ
ｉｎ Ｍｏ１ｅｃｕｌａｒ ＢｉｏｌｏｇｙＪ （
Ｓｐｒｉｎｇｃｒ−Ｖｅｒｌａｇ）などに記載されてい
る。“DHA C1” edited by H.GIOVeir
onina J ((RL Press), B, Pe
``^Practical'' written by rbal
Guide to Molecular CIonin
gJ (Wiley IntOrSCiOnCQ), R
, F, sch+ctr and P, C, Wensink [practtcatMethods
in Molecular BiologyJ (
Springcr-Verlag), etc.
（発明が解決しようとする問題点）
前記従来の方法は、いずれも、濾過フィルターを用いる
ことなく、遠心分離機によって沈澱と、上清に分離し、
上清を、新たな容器にデカンテーションによって取得し
たり、上清を排液して沈澱を取得したりしていた。この
ため、従来の方法は研究者の手をわずられし、長時間に
亘って、研究者を知的思考から遠ざけ、深夜にも作業を
継続しなくてはならない、バが多かった。(Problems to be Solved by the Invention) In all of the above conventional methods, a precipitate and a supernatant are separated by a centrifuge without using a filtration filter,
The supernatant was collected by decantation into a new container, or the supernatant was drained and the precipitate was collected. For this reason, conventional methods often require researchers to be involved, keep them away from intellectual thinking for long periods of time, and require them to continue working late into the night.
（問題点を解決するための手段）
本発明は、上記問題点を解決するために、遠心分離操作
をできる限り排除し、通常の執務時間内に全工程が終了
できるために、濾過を用いただけでなく、その結果とし
て、容易にラボラトリ−オートメーションを可能として
、原核細胞型粒子内の小型環状二本鎖サテライトＤＮＡ
を分取する方法を提供するものである。(Means for Solving the Problems) In order to solve the above problems, the present invention eliminates the centrifugation operation as much as possible and uses filtration to complete the entire process within normal office hours. but, as a result, small circular double-stranded satellite DNA within prokaryotic particles, allowing easy laboratory automation.
The present invention provides a method for preparative separation of .
（作用）
本発明による小型環状二本鎖サテライトＤＮＡを分取す
る方法の出発試料である、原核細胞型粒子の、水溶性不
純物を含む懸濁液には多様なものがあり、独立栄養原核
生物即ちラン藻類や、光合成細菌並びに化学合成細菌の
培養液は、上記水溶性不純物を含む懸濁液の一種である
が、これらは、菌体の他には無機塩類のみを含む、又は
、真核生物のミトコンドリアや、集緑体などは、その構
成から原核細胞型粒子と認められるが、これらの懸濁液
は、真核細胞を破砕し、その破砕された細胞から分取さ
れたもので、既に、【よぼ無機塩の溶液たる緩衝液に懸
濁されている。又従属栄養原核生物は、糖類やアミノ酸
、ビタミンなどの栄養素を含む培地中に懸濁されている
。この様に多様４１懸濁液に含まれている原核細胞型粒
子は、その種類によって、最小の径が知られているが、
その径よりも小さい孔径をもつ表面捕捉型濾過フィルタ
ーを用いて前記原核細胞型粒子の、水溶性不純物を含む
懸濁液を濾過すると、原核細胞型粒子は、前記濾過フィ
ルター１に残り、原核細胞を懸濁していた液は、濾過フ
ィルターを透過し、排除される。(Function) There are various types of suspensions containing water-soluble impurities of prokaryotic cell-type particles, which are the starting samples for the method of separating small circular double-stranded satellite DNA according to the present invention. In other words, the culture solutions of cyanobacteria, photosynthetic bacteria, and chemosynthetic bacteria are a type of suspension containing the above-mentioned water-soluble impurities; Mitochondria and plastids of organisms are recognized as prokaryotic cell-type particles due to their composition, but these suspensions are obtained by crushing eukaryotic cells and separating them from the crushed cells. It is already suspended in a buffer solution, which is a solution of an inorganic salt. Heterotrophic prokaryotes are also suspended in a medium containing nutrients such as sugars, amino acids, and vitamins. The minimum diameter of the prokaryotic cell-type particles contained in these various 41 suspensions is known depending on the type, but
When a suspension of the prokaryotic cell-type particles containing water-soluble impurities is filtered using a surface-trapping filter having a pore size smaller than that of the prokaryotic cell-type particles, the prokaryotic cell-type particles remain in the filtration filter 1, and the prokaryotic cell-type particles remain in the filtration filter 1. The suspended liquid passes through the filter and is removed.
この後、原核細胞型粒子を上に残した」−記濾過フイル
ターを、溶菌液中に浸すと原核細胞型粒子は破壊され、
水溶性成分は、溶菌液中に溶出づるが、この為に使われ
る溶菌液は、細胞壁をイＴす原核細胞型粒子には、まず
に細胞壁分解酵素液を用い、ｌ胸壁を消失させ、スフェ
ロプラストとなった原核細胞型粒子の細胞壁分解酵素液
に、アルカリ液を加え、細胞壁分解酵素を加水分解によ
って失活さＶると同時に、溶菌と、溶菌の結果溶出した
蛋白質成分を加水分解する。又細胞壁を有さない原核細
胞型粒子には、アルノｊり液のみが用いられる。After this, the prokaryotic particles were destroyed when the filter was immersed in the lysis solution, leaving the prokaryotic particles on top.
The water-soluble components are eluted into the lysate, but the lysate used for this purpose is to destroy the cell wall.For prokaryotic particles, a cell wall-degrading enzyme solution is first used to eliminate the thoracic wall, An alkaline solution is added to the cell wall-degrading enzyme solution of the prokaryotic cell-type particles that have become feroplasts, and the cell wall-degrading enzyme is inactivated by hydrolysis, and at the same time, bacteriolysis and protein components eluted as a result of bacteriolysis are hydrolyzed. . For prokaryotic cell-type particles that do not have a cell wall, only Arno lysate is used.
更に高塩濃度液を加える工程により、塩析可能な水溶性
成分を沈澱させるとともに、アルカリを中和する。この
工程によってＲＮＡとともに、高分子の染色体ＤＮＡは
スーパーコイル状態となり沈澱させることができる。史
に前記高塩濃度液によって生じた沈澱は濾過フィルター
によって濾過すると、透過液から除去される。最後の工
程で、エタノールを加えると、塩類の大部分はエタノー
ルに溶解するが、小型環状二本鎖ＤＮＡはエタノールに
よって沈澱するので、上清を除去すると、小型環状二本
鎖サテライトＤＮＡが、極めて少ない不純物とともに固
形にて分取される。Furthermore, by adding a high salt concentration solution, water-soluble components that can be salted out are precipitated and alkalis are neutralized. Through this step, high-molecular chromosomal DNA can be brought into a supercoiled state and precipitated together with RNA. The precipitate historically produced by the high salt concentration solution is removed from the permeate when it is filtered through a filtration filter. In the last step, when ethanol is added, most of the salts are dissolved in ethanol, but the small circular double-stranded DNA is precipitated by ethanol, so when the supernatant is removed, the small circular double-stranded satellite DNA is extremely It is collected in solid form with few impurities.
（実施例）
１〉常法により、培養したプラスミドｐＢＲ３２２を含
む大腸菌培養液５ｄをシリンジでとり、孔径０．２２Ｍ
、直径２５ａｍのポリビニリデン７０ライド製メンブレ
ンフイルターユニツトを上記シリンジに結合し、シリン
ジのピストンを押すことによって、培養液の内閲体を上
記メンブレンフィルターに取得し、培地を排出した。(Example) 1> Using a syringe, take 5 d of E. coli culture solution containing the cultured plasmid pBR322 using a conventional method, and prepare a pore size of 0.22M.
A polyvinylidene 70 Ride membrane filter unit having a diameter of 25 am was connected to the syringe, and by pushing the piston of the syringe, the culture solution was collected in the membrane filter and the culture medium was discharged.
２）一度取りはずしたシリンジに、５ＱｍＨグルコース
、 １０ｍＨＥＤＴＡ、 ２５ｎ＋ＨＴｒｉｓ−１１Ｃ
ｅ ＰＨ８゜０のＱ度に調製した緩衝液５−をとり、
再度、菌体を保持しているメンブレンフィルターユニッ
トを結合し、ピストンを押して、上記緩衝液に、菌体と
、その菌体を保持するメンブレンフィルターユニット内
部を洗浄せしめた。2) Add 5QmH glucose, 10mHEDTA, 25n+HTris-11C to the syringe once removed.
e Take the buffer solution 5- prepared to a Q degree of PH8゜0,
The membrane filter unit holding the bacterial cells was connected again, and the piston was pushed to allow the buffer solution to wash the bacterial cells and the inside of the membrane filter unit holding the bacterial cells.
３）洗浄された菌体を保持するメンブレンフィルターユ
ニットに結合したシリンジに、２■／Ｉｌｉ！ライソザ
イム、５ＱｉＨグルコース、１０１ＨＥＤＴΔ、 ２５
ｍＨＴｒｉｓ−ｌｌｃｅ Ｄｌ＋８ 、０に調製さ
れた、細胞壁分解酵素液０．２成をつめピストンで押し
出し、？ ４で２０分間保持し、更に０ 、２ Ｎ Ｎ
ａ０ｌｌ。3) Add 2■/Ili! to the syringe connected to the membrane filter unit that holds the washed bacterial cells. Lysozyme, 5QiH glucose, 101HEDTΔ, 25
mHTris-llce Dl+8, 0.2 composition of cell wall degrading enzyme solution prepared to 0 was squeezed and extruded with a piston. 4 for 20 minutes, then 0, 2 N N
a0ll.
１％ＳＤＳ溶液０．４−をつめたシリンジと交換、半量
をメンブレンフィルターユニットに対１ノて押し出し、
メンブレンフィルターユニットの、排液孔を、ラボフィ
ルムによってふさぎ、氷水中に５分間放置した。Replace the syringe with a syringe filled with 0.4-ml of 1% SDS solution, extrude half the amount into the membrane filter unit,
The drainage hole of the membrane filter unit was covered with lab film and left in ice water for 5 minutes.
４）２ｄのプラスチック試験管内に上記シリンジ及びメ
ンブレンフィルターユニット中の液を取り、更に０．３
ｄの、３Ｍ酢酸ナトリウム、酢酸ＰＨ４，８の溶液を流
すと、自沈を含む水溶液が得られた。4) Take the liquid in the syringe and membrane filter unit into a 2d plastic test tube, and add 0.3
When the solution of 3M sodium acetate and acetic acid pH 4,8 in step d was passed through, an aqueous solution containing scuttling was obtained.
５）上記自沈を含む水溶液を、新たな、前記と同様なメ
ンブレンフィルターユニットと、シリンジを用いて、新
たに２ｄのプラスデック試験管に濾液的０．５ｄをとっ
た。5) Using a new membrane filter unit similar to that described above and a syringe, 0.5 d of the aqueous solution containing the scuttling was taken into a new 2 d Plus Deck test tube.
６）前記濾液に（８ｍのエタノールを加λ、零下７０℃
のフリーザー中に３０分子ｆｉ冒し、セルロースミニカ
ラムを通過さｖＳＴＥ緩衝液１００成で溶出させた。こ
の溶出液は、エチジウムブロマイドで染色され、ＤＮＡ
と思われる色を呈した、更に、その一部を、制限酵素、
Ｔａｑ ｒで消化した摂、エチジウムブロマイドで染色
し、ｐＢＲ３２２を同様な処理をしたもの、制限消化を
行なわないでエヂジウムブロマイドで染色したものと同
時に、寒天電気泳動でパターンを確認したところ、上記
沈澱の水溶液を、Ｔａｑｌで消化したものど、ｐＢＲ３
２２をＴａｑ ｌで消化したものは、同じ位置にそれぞ
れ７木のバンドを呈し、制限消化を行なわなかった検体
には、一本のバンドのみが検出された。このことから、
本実施例によって得られた沈澱は、菌体中に３まれてい
た、ＤＢＲ３２２であった事が判定された。6) Add 8 m of ethanol to the filtrate at 70°C below zero.
Thirty molecules were loaded in a freezer, passed through a cellulose minicolumn, and eluted with 100ml of vSTE buffer. This eluate was stained with ethidium bromide and DNA
Furthermore, some of them were treated with restriction enzymes,
When the pattern was confirmed by agar electrophoresis, the precipitate was digested with Taqr, stained with ethidium bromide, treated with pBR322 in the same way, and stained with ethidium bromide without restriction digestion. The aqueous solution of pBR3 was digested with Taql.
22 digested with Taq I exhibited seven bands at the same positions, whereas only one band was detected in the sample that was not subjected to restriction digestion. From this,
It was determined that the precipitate obtained in this example was DBR322, which was present in the bacterial cells.
（効果）
以上述べたように本発明によれば、小型環状二本鎖サテ
ライトＩ） Ｎ Ａを繁雑な操作なく人手できるもので
ある。(Effects) As described above, according to the present invention, small cyclic double-stranded satellite I) NA can be prepared manually without complicated operations.
出願人 ピイコー電子工業株式会社
代理人 弁理士 最 上 務、；７゛゛゛）（
他１名） ′Applicant Piiko Electronics Industry Co., Ltd. Agent Patent Attorney Mogami; 7゛゛゛）(
1 other person) ′
Claims (3)
の様な、小型環状二本鎖サテライトＤＮＡを内部に含む
、原核細胞並びに真核細胞のミトコンドリアや集緑体の
様な原核細胞型粒子から、前記小型環状二本鎖サテライ
トＤＮＡを分取する方法に於て、 前記小型環状二本鎖サテライトＤＮＡを含む原核細胞型
粒子の、水溶性不純物を含む懸濁液から、濾過フィルタ
ーを用いて原核細胞型粒子を濾過フィルター上に残し、
懸濁液の上清を濾過によつて除去する第１の工程、 前記洗浄された、原核細胞型粒子を付着した濾過フィル
ターを、溶菌液中に浸し、原核細胞を破壊し、内部に含
まれる蛋白質を分解する第２の工程、 前記溶菌液に、高塩濃度液を加え、不純物を沈澱させる
第３の工程、 前記沈澱を含む液を前記の濾過フィルターとは別の濾過
フィルターで濾過し、濾液を取得する第４の工程、およ
び 前記濾液にエタノールを加え、小型環状二本鎖ＤＮＡを
沈澱させ、上清を除去する第５の工程、から成る、原核
細胞型粒子内の小型環状二本鎖サテライトＤＮＡを分取
する方法。(1) From prokaryotic cell-type particles such as mitochondria and plastids of prokaryotic cells and eukaryotic cells, which contain small circular double-stranded satellite DNA such as plasmids and replicative forms of viruses; In the method for separating circular double-stranded satellite DNA, prokaryotic cell-type particles are separated from a suspension containing water-soluble impurities of the prokaryotic cell-type particles containing the small circular double-stranded satellite DNA using a filtration filter. remain on the filter,
A first step of removing the supernatant of the suspension by filtration, immersing the washed filter with prokaryotic cell-type particles attached in a lysing solution to destroy the prokaryotic cells and remove the prokaryotic cells contained therein. a second step of decomposing proteins; a third step of adding a high salt concentration solution to the lysis solution to precipitate impurities; filtering the solution containing the precipitate with a filter different from the filtration filter; a fourth step of obtaining a filtrate; and a fifth step of adding ethanol to said filtrate to precipitate small circular double stranded DNA and removing the supernatant. Method for fractionating strand satellite DNA.
解酵素とＳＤＳを含み、かつ該酵素が活性を示し易い塩
類濃度と、ＰＨに調整され、後に、アルカリ溶液の添加
によって、ＰＨが高められる、特許請求の範囲第１項に
記載した原核細胞粒子の小型環状二本鎖サテライトＤＮ
Ａを分取する方法。(2) In the second step, the lysate initially contains a cell wall degrading enzyme and SDS, and is adjusted to a salt concentration and pH at which the enzyme is likely to exhibit activity, and later, by adding an alkaline solution, A small circular double-stranded satellite DN of a prokaryotic cell particle according to claim 1, which has an increased pH.
A method to separate A.
ルターとは別の濾過フィルター又はカラムにて濾し取り
、除去する、特許請求の範囲第１項及び第２項のいずれ
かの項記に記載した原核細胞型粒子内の小型環状二本鎖
サテライトＤＮＡを分取する方法。(3) In the fifth step, the supernatant is filtered and removed using a filter or column different from the filtration filter, according to any one of claims 1 and 2. A method for fractionating small circular double-stranded satellite DNA in prokaryotic particles as described in the above section.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61077853A JPS62236486A (en) | 1986-04-04 | 1986-04-04 | Method for separating small-sized cyclic double-stranded satellite dna in procaryotic cell particle |
| EP87302138A EP0240191A3 (en) | 1986-03-13 | 1987-03-12 | Method of isolating dna contained in a virus or cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61077853A JPS62236486A (en) | 1986-04-04 | 1986-04-04 | Method for separating small-sized cyclic double-stranded satellite dna in procaryotic cell particle |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62236486A true JPS62236486A (en) | 1987-10-16 |
Family
ID=13645618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61077853A Pending JPS62236486A (en) | 1986-03-13 | 1986-04-04 | Method for separating small-sized cyclic double-stranded satellite dna in procaryotic cell particle |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62236486A (en) |
-
1986
- 1986-04-04 JP JP61077853A patent/JPS62236486A/en active Pending
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