JP5600675B2 - 未処理rca産物 - Google Patents
未処理rca産物 Download PDFInfo
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Description
濃度100ng/μl(TE緩衝液)の超らせんDNAプラスミドpHygGFPを標準法によって調製する。2×R緩衝液(Tris:HCl 100mM、pH=8.2;KCl 150mM;MgCl2 20mM、TWEEN(登録商標)20 0.02%、DTT 2mM、dNTP 2 mM)2.5ml、P緩衝液(Tris:HCl 10mM、pH=8.2;EDTA 0.5mM;TWEEN(登録商標)20 0.01%、2つのホスホロチオエート結合を3’末端に含むランダムヘキサマー0.08mM)2.5ml、1mg/ml φ29 DNAポリメラーゼ0.1ml、及び超らせんpHygGFPプラスミド250ngを使用して、RCA反応を組み立てる。このRCA反応混合物を30℃で16時間インキュベートして等温DNA増幅を引き起こし、次いで65℃で20分間加熱して、DNAポリメラーゼを不活化する。
実施例1と同様の未処理RCA DNA産物を生成する代替法を使用することができる。ここでは、反応混合物が、
2×R緩衝液2.5ml;
チオエート化ランダムヘキサマー2.5ml(TE緩衝液中)(最終濃度は40μMプライマーである);
100mM dNTP 20μl;
1M DTT 5μl;
1M MgCl2 50μl;
1mg/ml φ29DNAポリメラーゼ 100μl;及び
pHygGFP超らせんプラスミド 250ng
を含む。
実施例1又は2の超らせんプラスミドDNAは、Qiagen社から販売されているEndoFree Plasmid Giga Kitを使用して以下のように調製することができる。
ポリエチレンイミン(PEI)は、プラスミドDNAを濃縮して、細胞の陰イオン性表面と相互作用する正荷電粒子にする。エンドサイトーシスによって細胞に入った後、このポリマーの高い電荷密度はリソソームを破裂させ、細胞質ゾル内へDNAを放出し、核内への移動を可能にする。
Claims (18)
- 生細胞に形質移入するためのRCA産物の生産方法であって、
二本鎖DNA(dsDNA)、一本鎖DNA(ssDNA)及びRNAからなる群から選択される1種以上の環状核酸鋳型を、1種以上のオリゴヌクレオチドプライマーであってその少なくとも一部分が上記環状核酸鋳型の一部分と相補的であるオリゴヌクレオチドプライマーと混合し、
上記オリゴヌクレオチドプライマーを上記環状核酸鋳型にアニールさせ、
アニールしたプライマーと鋳型に、1種以上のポリメラーゼと所定量のデオキシリボヌクレオチド三リン酸(dNTP)を添加し、
上記環状核酸鋳型をRCAで複製してRCA産物を得て、
未処理のRCA産物を形質移入媒体に直接移す
ことを含む方法。 - 前記DNAポリメラーゼが3’−5’エキソヌクレアーゼ活性又は鎖置換活性を示す、請求項1記載の方法。
- 前記DNAポリメラーゼが、バクテリオファージφ29 DNAポリメラーゼ、Tts DNAポリメラーゼ、ファージM2 DNAポリメラーゼ、DNAポリメラーゼIのクレノウ断片、T5 DNAポリメラーゼ、PRD1 DNAポリメラーゼ、T4 DNAポリメラーゼホロ酵素、T7 DNAポリメラーゼ及びBst DNAポリメラーゼからなる群から選択される、請求項1記載の方法。
- 前記DNAポリメラーゼが3’−5’エキソヌクレアーゼ活性を示さない、請求項1記載の方法。
- 前記DNAポリメラーゼが、Taqポリメラーゼ、Tflポリメラーゼ、Tthポリメラーゼ、真核生物DNAポリメラーゼα、及び3’−5’エキソヌクレアーゼ活性がなくなるように改質されたDNAポリメラーゼからなる群から選択される、請求項4記載の方法。
- 前記dNTPの少なくとも一部分が、ホスホチオエート化ヌクレオチド、ロックド核酸(LNA)、dUTP、dITP、rNTP、5−メチルdCTP、2−アミノ−dATP、2−チオ−dTTP、4’−チオ−dTTP、4’−チオ−dCTP及びデアザ−dGTPからなる群から選択される修飾ヌクレオチドである、請求項1乃至請求項5のいずれか1項記載の方法。
- 前記環状核酸鋳型が、1以上のプロモーター配列と1以上のターゲット配列と1以上の終結配列とを含んでいる、請求項1乃至請求項6のいずれか1項記載の方法。
- 前記プロモーター配列及び終結配列が真核生物の配列である、請求項7記載の方法。
- 前記ターゲット配列が、宿主生物で免疫応答を誘発することのできる発現産物をコードする、請求項7又は請求項8記載の方法。
- 前記ターゲット配列が、タンパク質、メッセンジャーRNA(mRNA)配列、非コードRNA配列、マイクロRNA(miRNA)配列、低分子干渉RNA(siRNA)配列及びモノクローナル抗体(mAb)鎖の少なくともいずれかをコードする、請求項9記載の方法。
- 前記ターゲット配列が、細菌、ウイルス、真菌、寄生生物又は非寄生生物のうちの1種以上に由来する、請求項9又は請求項10記載の方法。
- 前記環状DNA鋳型が、各々宿主生物で免疫応答を誘発することのできる異なる発現産物をコードする2種以上のターゲット配列を含む、請求項1乃至請求項11のいずれか1項記載の方法。
- インビトロでの細胞の形質移入方法であって、
ホスホチオエート化ヌクレオチド、ロックド核酸(LNA)、dUTP、dITP、rNTP、5−メチルdCTP、2−アミノ−dATP、2−チオ−dTTP、4’−チオ−dTTP、4’−チオ−dCTP及びデアザ−dGTPからなる群から選択される修飾ヌクレオチドを含む未処理RCA産物を得る段階と、
未処理RCA産物を1以上の細胞に形質移入する段階と
を含む方法。 - 生物への投与に適した1種以上の未処理RCA産物を含むワクチンであって、前記RCA産物が、二本鎖DNA(dsDNA)、一本鎖DNA(ssDNA)及びRNAからなる群から選択される1以上の核酸鋳型から少なくとも部分的に生産される、ワクチン。
- 前記未処理RCA産物が、ホスホチオエート化ヌクレオチド、ロックド核酸(LNA)、dUTP、dITP、rNTP、5−メチルdCTP、2−アミノ−dATP、2−チオ−dTTP、4’−チオ−dTTP、4’−チオ−dCTP及びデアザ−dGTPからなる群から選択される修飾ヌクレオチドを含む、請求項14記載のワクチン。
- 前記未処理RCA産物が、生物で免疫応答を誘発することのできる発現産物をコードする1種以上のターゲット配列を含む、請求項14記載のワクチン。
- 生細胞の形質移入に適した1種以上の未処理RCA産物を含む形質移入製品であって、前記RCA産物が、二本鎖DNA(dsDNA)、一本鎖DNA(ssDNA)及びRNAからなる群から選択される1以上の核酸鋳型から少なくとも部分的に生産される、形質移入製品。
- 前記未処理RCA産物が、ホスホチオエート化ヌクレオチド、ロックド核酸(LNA)、dUTP、dITP、rNTP、5−メチルdCTP、2−アミノ−dATP、2−チオ−dTTP、4’−チオ−dTTP、4’−チオ−dCTP及びデアザ−dGTPからなる群から選択される修飾ヌクレオチドを含む、請求項17記載の形質移入製品。
Applications Claiming Priority (3)
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US12/169,993 US20100008939A1 (en) | 2008-07-09 | 2008-07-09 | Unprocessed rolling circle amplification product |
US12/169,993 | 2008-07-09 | ||
PCT/SE2009/050667 WO2010005365A1 (en) | 2008-07-09 | 2009-06-04 | Unprocessed rolling circle amplification product |
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JP2011527570A5 JP2011527570A5 (ja) | 2012-05-31 |
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US (1) | US20100008939A1 (ja) |
EP (1) | EP2307575B1 (ja) |
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US20110195457A1 (en) * | 2010-02-09 | 2011-08-11 | General Electric Company | Isothermal amplification of nucleic acid using primers comprising a randomized sequence and specific primers and uses thereof |
US9644232B2 (en) * | 2013-07-26 | 2017-05-09 | General Electric Company | Method and device for collection and amplification of circulating nucleic acids |
JP6080884B2 (ja) | 2014-03-28 | 2017-02-15 | 富士フイルム株式会社 | 重合性化合物、ポリマー、重合性組成物、フィルム、および投映像表示用ハーフミラー |
WO2016170179A1 (en) * | 2015-04-24 | 2016-10-27 | Qiagen Gmbh | Method for immobilizing a nucleic acid molecule on solid support |
WO2016170182A1 (en) | 2015-04-24 | 2016-10-27 | Qiagen Gmbh | Method for immobilizing a nucleic acid molecule on a solid support |
WO2016187359A1 (en) * | 2015-05-18 | 2016-11-24 | Scott Schaffer | Clip-on nasal air humidifying and epistaxis-prevention device and methods for use with supplemental oxygen |
US10077459B2 (en) * | 2016-05-04 | 2018-09-18 | General Electric Company | Cell-free protein expression using rolling circle amplification product |
US10350307B2 (en) * | 2017-09-18 | 2019-07-16 | General Electric Company | In vivo RNA or protein expression using double-stranded concatemeric DNA including phosphorothioated nucleotides |
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US5714320A (en) * | 1993-04-15 | 1998-02-03 | University Of Rochester | Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides |
US7030098B2 (en) * | 1999-03-12 | 2006-04-18 | The Board Of Trustees Of The Leland Stanford Junior University | DNA vaccination for treatment of autoimmune disease |
AU2001238535A1 (en) * | 2000-02-16 | 2001-08-27 | Large Scale Biology Corporation | Rolling circle replicon expression vectors |
US6323009B1 (en) * | 2000-06-28 | 2001-11-27 | Molecular Staging, Inc. | Multiply-primed amplification of nucleic acid sequences |
CU23400A1 (es) * | 2003-04-29 | 2009-08-04 | Ct Ingenieria Genetica Biotech | Método para la obtención de minicírculos replicativos de adn para transferencia génica o como inmunomoduladores |
US20050202453A1 (en) * | 2003-05-28 | 2005-09-15 | Wen-Yuan Song | Nucleic acid amplification in yeast |
AU2005314431B2 (en) * | 2004-12-11 | 2011-02-17 | Cytogenix, Inc. | Cell free biosynthesis of high-quality nucleic acid and uses thereof |
CN101103122A (zh) * | 2004-12-11 | 2008-01-09 | 西托吉尼克斯公司 | 高质量核酸的无细胞生物合成及其用途 |
WO2007018744A2 (en) * | 2005-08-03 | 2007-02-15 | Cytogenix, Inc. | Cell-free biosynthesis of nucleic acid |
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2008
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- 2009-06-04 EP EP09794720.4A patent/EP2307575B1/en active Active
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EP2307575A1 (en) | 2011-04-13 |
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WO2010005365A1 (en) | 2010-01-14 |
EP2307575A4 (en) | 2012-09-12 |
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