JP5203960B2 - 短鎖核酸の濃縮方法 - Google Patents
短鎖核酸の濃縮方法 Download PDFInfo
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- JP5203960B2 JP5203960B2 JP2008543848A JP2008543848A JP5203960B2 JP 5203960 B2 JP5203960 B2 JP 5203960B2 JP 2008543848 A JP2008543848 A JP 2008543848A JP 2008543848 A JP2008543848 A JP 2008543848A JP 5203960 B2 JP5203960 B2 JP 5203960B2
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Classifications
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- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
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Description
(α2)上記核酸(α1)とは異なる少なくとも1種の成分を含有する好ましくは水性の流動体相P1を提供すること;
ii)相P1を陰イオン交換マトリックスに接触させて核酸(α1)を陰イオン交換マトリックスに結合させること;
iii)洗浄緩衝液で陰イオン交換マトリックスを任意で洗浄し、その際、核酸(α1)は陰イオン交換マトリックスに結合したままであること;並びに
iv)前記陰イオン交換マトリックスから陰イオン交換マトリックスに結合した核酸(α1)を取り出し、好ましくは溶出して、核酸(α1)を含有する好ましくは水性の流動体相P2を得ること。
I)細胞を提供すること、
II)細胞を溶解して細胞溶解物を得ること、及び
III)細胞溶解物から核酸(α1)とは異なる成分(α2)を少なくとも1種を少なくとも部分的に任意で分離することの方法工程を含む方法によって入手可能な細胞溶解物である。
0.1〜1モル/Lの、特に好ましくは0.25〜0.75モル/Lの、最も好ましくは0.4〜0.6モル/LのNaCl及び0.1〜10%(v/v)の、特に好ましくは0.5〜5%(v/v)の、最も好ましくは0.75〜1.5%(v/v)のトリトンX−100を含有し、6〜8の範囲内の、特に好ましくは約7のpHを有する緩衝液、
又は
0.5〜10モル/Lの、特に好ましくは1〜5モル/Lの、最も好ましくは1.5〜3モル/Lのグアニジニウムイソチオシアネート、1〜50ミリモル/Lの、特に好ましくは5〜40ミリモル/Lの、最も好ましくは10〜20ミリモル/Lのクエン酸ナトリウム及び10〜60%(v/v)の、特に好ましくは20〜50%(v/v)の、最も好ましくは30〜40%(v/v)のエタノールを含有し、6〜8の範囲内の、特に好ましくは約7のpHを有する緩衝液である。
(β1)溶解緩衝液又は溶解緩衝液濃縮物
(β2)陰イオン交換マトリックス
(β3)溶出緩衝液
(β4)任意で浮遊緩衝液
(β5)任意で中和緩衝液
(β6)任意で洗浄緩衝液、及び
(β7)任意で抽出用溶剤、たとえば、フェノール、エタノールのようなアルコール又はそれらの混合物を含む。
(γ1)最初に記載された精製方法に従って、300ヌクレオチド未満の、好ましくは200ヌクレオチド未満の、特に好ましくは100ヌクレオチド未満の、さらに好ましくは50ヌクレオチド未満の、最も好ましくは、25ヌクレオチド未満の長さを持つ核酸を濃縮することを含む診断方法によって疾患を診断すること、及び
(γ2)診断された疾患を治療的に処置すること。
106個のJurkat細胞を1μgのmiR177アンチセンスmiRNAと混合し、0.5MのNaCl、1%(v/v)のトリトンX−100を含有する溶解緩衝液550μLの助けを借りて細胞を溶解した。氷上で10分間インキュベートした後、550μLの酸性フェノールを添加した。ボルテックスで撹拌し、次いで20 800xgにて5分間遠心して水性相を取り出し、ポリエチレンイミンで被覆した磁性粒子652μgと混合した。
106個のJurkat細胞を1μgのlet7aアンチセンスRNAと混合し、実施例1で特定されたように溶解し、磁性粒子に結合させた。水による洗浄工程の後、20μLの緩衝液で溶出を行ったが、100ミリモル/LのNaCl、250ミリモル/LのNaCl、400ミリモル/LのNaCl、100ミリモル/LのKCl、250ミリモル/LのKCl及び400ミリモル/LのKClが、溶出緩衝液として使用された。等分の溶出物を15%のアクリルアミドゲルに適用し、硝酸銀で染色した(図2)。
溶出緩衝液として10〜100ミリモル/LのMgCl2を含有する緩衝液を用いて、手順は実施例2のとおりであった。等分の溶出物を15%のアクリルアミドゲルに適用し、硝酸銀で染色した(図3)。
溶出緩衝液として10〜85ミリモル/LのCaCl2を含有する緩衝液用いて、手順は実施例2のとおりであった。等分の溶出物を15%のアクリルアミドゲルに適用し、硝酸銀で染色した(図4)。
25〜400ミリモル/Lの硫酸アンモニウム又は25〜400ミリモル/Lの塩化アンモニウムを含有する緩衝液用いて、手順は実施例2のとおりであった。等分の溶出物を15%のアクリルアミドゲルに適用し、硝酸銀で染色した(図5)。
Claims (22)
- 300ヌクレオチド未満の長さを持つ核酸を、300ヌクレオチド未満の長さを持つ核酸以外に他の成分を含む複雑な生物学的組成物から、濃縮する方法であって、
i)下記の成分を含有する水性相P1を提供する
(α1)少なくとも1種の300ヌクレオチド未満の長さを持つ核酸、及び
(α2)前記核酸(α1)とは異なる少なくとも1種の成分、
ii)相P1を陰イオン交換マトリックスに接触させて核酸(α1)を陰イオン交換マトリックスに結合させるが、このとき核酸が、3〜7の範囲のpH値で陰イオン交換マトリックスへ結合し、陰イオン交換マトリックスが、水性相P 1 が陰イオン交換マトリックスと接触する条件下では、少なくとも部分的には陽イオン形態の官能基を有し、かつ陰イオン交換マトリックスが磁性粒子上のコーティングの形態で存在し、
iii)洗浄緩衝液によって陰イオン交換マトリックスを洗浄し、その際、核酸(α1)は陰イオン交換マトリックスに結合したままである、及び
iv)陰イオン交換マトリックスに結合した核酸(α1)を前記陰イオン交換マトリックスから溶出して核酸(α1)を含有する水性相P 2を得る方法工程を含む前記方法。 - 核酸(α1)がRNAである、請求項1に記載の方法。
- RNAが、miRNA、プレ−miRNA、siRNA、snRNA、snoRNA、tRNA、5S−rRNA、5.8S−rRNA、又はそれらからの少なくとも2つの混合物を含む群から選択される、請求項2に記載の方法。
- RNAが、miRNA、プレ−miRNA、tRNA又はmiRNAとtRNAの混合物である請求項2に記載の方法。
- 陰イオン交換マトリックスが、アミノ基、ホスフィン基、ヒドラジン基及びイミン基を含む群から選択される、請求項1〜4のいずれかに記載の方法。
- 溶出が、陰イオン交換マトリックスと、陰イオン交換マトリックスの官能基と核酸(α1)との間の結合を溶解する溶出緩衝液とを接触させることにより実施され、それにより核酸(α1)を含む水性相P 2 として溶離され、このとき溶出緩衝液が、水性塩溶液である、請求項1〜5のいずれかに記載の方法。
- 溶出緩衝液が、アルカリ金属ハロゲン化物、アルカリ土類金属ハロゲン化物、アンモニウム塩又はこれらの塩の少なくとも2つの混合物であり、それにより溶出緩衝液が、緩衝システムを含むことができる、請求項5に記載の方法。
- 溶出緩衝液が、水溶性カルシウム塩、水溶性マグネシウム塩、水溶性アンモニウム塩又はこれらの塩の少なくとも2つの混合物を含有する、請求項6又は7に記載の方法。
- 溶出緩衝液が塩化カルシウムを含有し、かつ下記の特徴の1つを有する請求項6〜8のいずれかに記載の方法:
a)CaCl 2 が、1〜1000mmol/lの範囲の濃度で存在する、
b)CaCl 2 が、5〜500mmol/lの範囲の濃度で存在する、
c)CaCl 2 が、10〜100mmolの範囲の濃度で存在する。 - 溶出緩衝液が塩化マグネシウムを含有し、かつ下記の特徴の一つを有する、請求項6〜8のいずれかに記載の方法:
a)MgCl 2 が、1〜1000mmol/lの範囲の濃度で存在する、
b)MgCl 2 が、5〜500mmol/lの範囲の濃度で存在する、
c)MgCl 2 が、10〜100mmolの範囲の濃度で存在する。 - 溶出緩衝液が硫酸アンモニウム又は塩化アンモニウムを含有し、かつ下記の特徴の一つを有する、請求項6〜8のいずれかに記載の方法
a)該塩が、1〜1000mmol/lの範囲の濃度で存在する、
b)該塩が、5〜500mmol/lの範囲の濃度で存在する、
c)該塩が、20〜300mmolの範囲の濃度で存在する。 - 溶出緩衝液のpH値が、5〜12の範囲にある、請求項1〜11のいずれかに記載の方法。
- 専らカルシウム塩、及び/又はアンモニウム塩を含む溶出緩衝液が用いられる、請求項1〜6のいずれかに記載の方法。
- 水、及び60mmol/l以下の濃度のCaCl 2 からなる、又は水、及び170〜200mmol/l以下の濃度の硫酸アンモニウム若しくは塩化アンモニウムからなる溶出緩衝液を用いる、請求項13に記載の方法。
- 溶出緩衝液が下記から選択される、請求項1〜6のいずれかに記載の方法:
a)水に溶解された、
i)1〜10000mmol/l、10〜5000mmol/l又は50〜1000mmol/lのトリス、
ii)1〜1000mmol/l、5〜800mmol/l又は10〜500mmol/lのアルカリ金属塩、
iii)1〜400mmol/l、10〜300mmol/l又は50〜200mmol/lのアンモニウム塩、及び
iv)0.1〜200mmol/l、0.5〜100mmol/l又は1〜50mmol/lのマグネシウム塩
を含む、溶出緩衝液EP 1 ;
b) 水に溶解された、1〜1000mmol/l、5〜500mmol/l又は10 〜100mmol/lのマグネシウム塩を含む、溶出緩衝液EP 2 ;
c) 水に溶解された、1〜1000mmol/l、5〜500mmol/l又は10〜100mmol/lのカルシウム塩を含む溶出緩衝液EP 3 ;
d) 水に溶解された、1〜1000mmol/l、5〜500mmol/l又は20 〜300mmol/lのアンモニウム塩を含む溶出緩衝液EP 4 ;
e) 水に溶解された、1〜2000mmol/l、10〜1000mmol/l又は 100〜500mmol/lのアルカリ金属塩を含む溶出緩衝液EP 5 。 - 方法工程i)で提供される水性相P1が細胞溶解物であり、
このとき細胞溶解物が、
I)細胞を提供する、
II)細胞を溶解して細胞溶解物を得る、及び
III)該細胞溶解物から核酸(α1)とは異なる少なくとも1種の成分(α2)を少なくとも部分的に分離する
方法工程を含む方法によって入手可能である、請求項1〜15のいずれかに記載の方法。 - 相P2におけるRNAの総量に対する相P2における300ヌクレオチド未満の長さを持つRNAの相対量が、相P1におけるRNAの総量に対する相P1における300ヌクレオチド未満の長さを持つRNAの相対量よりも、少なくとも2倍多いか、又は
水性相P2におけるmiRNAとtRNAの総量に対する水性相P2におけるmiRNAの相対量が、水性相P1におけるmiRNAとtRNAの総量に対する水性相P1におけるmiRNAの相対量よりも、少なくとも2倍多い、請求項2〜16のいずれかに記載の方法。 - 核酸(α1)が、アルカリ金属塩の存在下、方法工程ii)における陰イオン交換マトリックスへ結合され、結合中のアルカリ金属塩の濃度が、0.01〜10mol/lの範囲内にある、請求項1〜17のいずれかに記載の方法。
- 300ヌクレオチド未満の長さを持つ核酸を濃縮するためのキットであって、
(β1)溶解緩衝液又は溶解緩衝液濃縮物
(β2)陰イオン交換マトリックスであって、このとき陰イオン交換マトリックスが磁性粒子上のコーティングの形態で存在し、
(β3)下記から選択される、溶出緩衝液:
(β3.1)水に溶解された、
i)1〜10000mmol/l、10〜5000mmol/l又は50〜1000mmol/lのトリス、
ii)1〜1000mmol/l、5〜800mmol/l又は10〜500mmol/lのアルカリ金属塩、
iii)1〜400mmol/l、10〜300mmol/l又は50〜200mmol/lのアンモニウム塩、及び
iv)0.1〜200mmol/l、0.5〜100mmol/l又は1〜50mmol/lのマグネシウム塩
を含む、溶出緩衝液EP 1 ;
(β3.2)水に溶解された、1〜1000mmol/l、5〜500mmol/l又は10〜100mmol/lのマグネシウム塩を含む溶出緩衝液EP 2 ;
(β3.3)水に溶解された、1〜1000mmol/l、5〜500mmol/l又は10〜100mmol/lのカルシウム塩を含む溶出緩衝液EP 3 ;
(β3.4)水に溶解された、1〜1000mmol/l、5〜500mmol/l又は20〜300mmol/lのアンモニウム塩を含む溶出緩衝液EP 4 ;
(β3.5)水に溶解された、1〜2000mmol/l、10〜1000mmol/l又は100〜500mmol/lのアルカリ金属塩を含む溶出緩衝液EP 5 。 - 緩衝液EP 1 が7〜11の範囲のpH値を有し;緩衝液EP 2 が6〜10の範囲のpH値を有し;緩衝液EP 3 が6〜10の範囲のpH値を有し;緩衝液EP 4 が6〜10の範囲のpH値を有し;緩衝液EP 5 が6〜10の範囲のpH値を有する、請求項19に記載のキット。
- 陰イオン交換マトリックス(β2)が、アミノ基、ホスフィン基、ヒドラジン基及びイミン基を含む群から選択される官能基を有する請求項19又は20に記載のキット。
- 請求項1〜18のいずれかに記載の方法における請求項19〜21のいずれかに記載のキットの使用。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2022071648A1 (ko) * | 2020-09-29 | 2022-04-07 | 주식회사 에이아이더뉴트리진 | 미생물 농축 또는 핵산 추출용 조성물 및 이를 이용한 미생물 농축 또는 핵산 추출 방법 |
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EP1960522B1 (de) | 2016-03-02 |
US20090081802A1 (en) | 2009-03-26 |
CN103173436A (zh) | 2013-06-26 |
AU2006323954A1 (en) | 2007-06-14 |
EP3064583A1 (de) | 2016-09-07 |
WO2007065950A1 (de) | 2007-06-14 |
CN101326284A (zh) | 2008-12-17 |
US7977109B2 (en) | 2011-07-12 |
AU2006323954B2 (en) | 2012-05-10 |
ES2572630T3 (es) | 2016-06-01 |
EP3524678A1 (de) | 2019-08-14 |
DE102005059315A1 (de) | 2007-06-14 |
CN105018470A (zh) | 2015-11-04 |
JP2009518020A (ja) | 2009-05-07 |
EP1960522A1 (de) | 2008-08-27 |
ES2729164T3 (es) | 2019-10-30 |
EP3064583B1 (de) | 2019-03-27 |
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