ES2638853T3 - Amplificación isotérmica de ácido nucleico - Google Patents
Amplificación isotérmica de ácido nucleico Download PDFInfo
- Publication number
- ES2638853T3 ES2638853T3 ES09762016.5T ES09762016T ES2638853T3 ES 2638853 T3 ES2638853 T3 ES 2638853T3 ES 09762016 T ES09762016 T ES 09762016T ES 2638853 T3 ES2638853 T3 ES 2638853T3
- Authority
- ES
- Spain
- Prior art keywords
- target molecule
- nucleic acid
- primer
- molecule
- oligonucleotide
- 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.)
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- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Un procedimiento isotérmico para amplificar una molécula objetivo de ácido nucleico bicatenario que comprende las siguientes etapas: (a) proporcionar: (i) cebadores en dirección 5' y en dirección 3', comprendiendo cada uno una molécula de ADN monocatenario de menos de 30 nucleótidos, al menos una porción de la cual es complementaria a una secuencia de la molécula objetivo; (ii) un oligonucleótido que comprende una molécula de ADN monocatenario de al menos 30 nucleótidos, al menos una porción de la cual es complementaria a una secuencia de la molécula objetivo que interviene en los cebadores directo e inverso, en donde el oligonucleótido tiene un extremo terminal 3'; (b) poner en contacto el oligonucleótido (ii) con la recombinasa para permitir que invada la región complementaria de la molécula objetivo, volviendo de este modo la región complementaria de la molécula objetivo y regiones adyacentes monocatenarias; (c) aplicar el cebador en dirección 5' a la región monocatenaria de la molécula objetivo y extender el extremo terminal 3' del cebador en dirección 5' con polimerasa y dNTP para producir una molécula objetivo de ácido nucleico bicatenario; (d) aplicar el cebador en dirección 3' a la molécula objetivo monocatenaria y extender el extremo terminal 3' del cebador en dirección 3' con polimerasa y dNTP para producir otra molécula objetivo de ácido nucleico bicatenario; (e) continuar la reacción mediante la repetición de (b) hasta (d).
Description
Sacarosa fosforilasa (Sigma S0937) Se disolvió hasta 0,4 u/µL en glicerol al 40%/H2O UvsX, UvsY; 100 µM en acetato de K 300 mM; glicerol al 50%. Gp32 (NEB, 10 mg/mL) Klenow, exo -(Jena Biosciences, 50 u/µL) utilizado a una concentración final de 0,05 u/µL
- Componente
- Concentración final de reacción
- Tris pH 8
- 10 mM
- Acetato de Mg
- 10 mM
- BSA**
- 0,1 mg/mL
- DTT**
- 5 mM
- DMSO**
- 5%
- PEG 1000**
- 5%
- Sacarosa**
- 150 mM
- ATP
- 2mM
- dNTP
- 200 µM
- Sybr Green*
- 1:100.000
- Oligonucleótidos
- Como se muestra en los ejemplos
- gp32
- 0,5 µM
- Fosfocreatina (diTRIS) pH hasta 7,8 con KOH
- 75 mM
- Creatina Quinasa
- 1µM
- Mioquinasa**
- 1µM
- Pirofosfatasa**
- 1µM
- UvsY
- 1,5 µM
- UvsX
- 1,5 µM
- Sacarosa fosforilasa**
- 1µM
- Klenow
- 0,1µM
- ADN plantilla
- Como se muestra en los ejemplos
- ** = componentes que se encuentra que optimizan, pero que no son esenciales para la amplificación.
Se añadió plantilla de ensayo a una mezcla de los componentes de reacción excepto por UvsX y Klenow. Los componentes de reacción se incubaron con una muestra de ensayo durante 5 minutos a la temperatura de trabajo (40° C) y se añadieron UvsX y Klenow. Los volúmenes totales de muestra fueron de 20 µL colocados en placas de
10 microtitulación de 384 pocillos de bajo volumen. La fluorescencia se evaluó en un BMG-fluostar-II. La fluorescencia se controló a intervalos de un minuto por excitación a 480 nm y leyendo la emisión a 520 nm para la fluorescencia Sybr Green (a menos que se indique lo contrario).
Ejemplo 1: Sistema de dos cebadores, sin oligonucleótido intermedio (sistema de la técnica anterior)
El protocolo utilizado es el mismo que aquel descrito anteriormente a menos que se indique lo contrario. Los 15 constituyentes de oligonucleótidos comprendían dos cebadores a una concentración final de 150 nM junto con la
16
Claims (1)
-
imagen1
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0810650A GB0810650D0 (en) | 2008-06-11 | 2008-06-11 | Strand invasion based isothermal nucleic acid amplification |
GB0810650 | 2008-06-11 | ||
GB0822533A GB0822533D0 (en) | 2008-12-11 | 2008-12-11 | Strand invasion based isothermal nucleic acid amplifications (2) |
GB0822533 | 2008-12-11 | ||
PCT/GB2009/050662 WO2009150467A1 (en) | 2008-06-11 | 2009-06-11 | Isothermal nucleic acid amplification |
Publications (1)
Publication Number | Publication Date |
---|---|
ES2638853T3 true ES2638853T3 (es) | 2017-10-24 |
Family
ID=40872336
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ES09762016.5T Active ES2638853T3 (es) | 2008-06-11 | 2009-06-11 | Amplificación isotérmica de ácido nucleico |
Country Status (16)
Country | Link |
---|---|
US (3) | US9062344B2 (es) |
EP (3) | EP3249056B1 (es) |
JP (2) | JP5798917B2 (es) |
CN (2) | CN104862385B (es) |
CA (1) | CA2727212C (es) |
CY (2) | CY1119346T1 (es) |
DK (2) | DK2660336T3 (es) |
ES (1) | ES2638853T3 (es) |
HR (2) | HRP20171286T1 (es) |
HU (2) | HUE034561T2 (es) |
LT (2) | LT2304054T (es) |
PL (2) | PL2304054T3 (es) |
PT (2) | PT2304054T (es) |
SI (2) | SI2660336T1 (es) |
WO (1) | WO2009150467A1 (es) |
ZA (1) | ZA201008749B (es) |
Families Citing this family (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8293684B2 (en) * | 2006-11-29 | 2012-10-23 | Exiqon | Locked nucleic acid reagents for labelling nucleic acids |
EP3249056B1 (en) | 2008-06-11 | 2019-04-10 | GeneForm Technologies Limited | Method of atp regeneration in a recombinase reaction |
US9309557B2 (en) | 2010-12-17 | 2016-04-12 | Life Technologies Corporation | Nucleic acid amplification |
US9334531B2 (en) | 2010-12-17 | 2016-05-10 | Life Technologies Corporation | Nucleic acid amplification |
US9309566B2 (en) | 2010-12-17 | 2016-04-12 | Life Technologies Corporation | Methods, compositions, systems, apparatuses and kits for nucleic acid amplification |
KR101963462B1 (ko) | 2010-10-04 | 2019-03-28 | 제납시스 인크. | 재사용 가능한 자동화 평행 생물 반응 시스템 및 방법 |
US9399217B2 (en) | 2010-10-04 | 2016-07-26 | Genapsys, Inc. | Chamber free nanoreactor system |
US9184099B2 (en) | 2010-10-04 | 2015-11-10 | The Board Of Trustees Of The Leland Stanford Junior University | Biosensor devices, systems and methods therefor |
JP5907990B2 (ja) | 2010-12-17 | 2016-05-11 | ライフ テクノロジーズ コーポレーション | 核酸増幅のための方法、組成物、システム、装置およびキット |
US8585973B2 (en) | 2011-05-27 | 2013-11-19 | The Board Of Trustees Of The Leland Stanford Junior University | Nano-sensor array |
US9926596B2 (en) | 2011-05-27 | 2018-03-27 | Genapsys, Inc. | Systems and methods for genetic and biological analysis |
CN106591103B (zh) | 2011-12-01 | 2021-06-04 | 吉纳普赛斯股份有限公司 | 用于高效电子测序与检测的系统和方法 |
CN102816756B (zh) * | 2012-09-07 | 2014-04-16 | 江苏奇天基因生物科技有限公司 | 等温核酸扩增反应试剂及等温核酸扩增方法 |
MX365323B (es) | 2013-03-15 | 2019-05-29 | Theranos Ip Co Llc | Amplificación de ácidos nucléicos. |
WO2014145298A1 (en) | 2013-03-15 | 2014-09-18 | Theranos, Inc. | Nucleic acid amplification |
WO2014152625A1 (en) | 2013-03-15 | 2014-09-25 | Genapsys, Inc. | Systems and methods for biological analysis |
US10450595B2 (en) | 2013-03-15 | 2019-10-22 | Theranos Ip Company, Llc | Nucleic acid amplification |
AU2014233145A1 (en) | 2013-03-15 | 2015-09-17 | Theranos Ip Company, Llc | Nucleic acid amplification |
KR20160036528A (ko) * | 2013-04-25 | 2016-04-04 | 오리온 디아그노스티카 오와이 | 가닥―침입 기반된 dna 증폭 방법 |
EP3042208A4 (en) | 2013-09-06 | 2017-04-19 | Theranos, Inc. | Systems and methods for detecting infectious diseases |
WO2015075198A1 (en) | 2013-11-22 | 2015-05-28 | Orion Diagnostica Oy | Detection of nucleic acids by strand invasion based amplification |
US10125393B2 (en) | 2013-12-11 | 2018-11-13 | Genapsys, Inc. | Systems and methods for biological analysis and computation |
US9822401B2 (en) | 2014-04-18 | 2017-11-21 | Genapsys, Inc. | Methods and systems for nucleic acid amplification |
GB201410022D0 (en) * | 2014-06-05 | 2014-07-16 | Orion Diagnostica Oy | Method |
US11268117B2 (en) | 2016-06-10 | 2022-03-08 | Life Technologies Corporation | Methods and compositions for nucleic acid amplification |
CN116397014A (zh) | 2016-07-20 | 2023-07-07 | 测序健康公司 | 用于核酸测序的系统和方法 |
US10443095B2 (en) * | 2016-08-02 | 2019-10-15 | Roche Molecular Systems, Inc. | Helper oligonucleotide for improved efficiency of amplification and detection/quantitation of nucleic acids |
WO2018038232A1 (ja) * | 2016-08-24 | 2018-03-01 | 国立大学法人東北大学 | 標的核酸の増幅産物の生産方法及びその利用 |
GB201618035D0 (en) | 2016-10-25 | 2016-12-07 | Orion Diagnostica Oy | Method |
CN107058287B (zh) * | 2017-01-16 | 2020-06-16 | 浙江大学 | 一种恒温扩增体系中生成单链产物的方法 |
EP3684951A4 (en) | 2017-09-21 | 2021-06-16 | Genapsys, Inc. | NUCLEIC ACID SEQUENCING SYSTEMS AND METHODS |
CN107893103A (zh) * | 2017-11-29 | 2018-04-10 | 默禾医疗科技(上海)有限公司 | 重组酶聚合酶扩增中重组酶和蛋白浓度比及活性定量法 |
EP3530755B1 (de) | 2018-02-26 | 2020-08-26 | AGCT GmbH | Verfahren zum anzeigen des fortschrittes der amplifikation von nukleinsäuren und kit zu dessen durchführung |
US20200224260A1 (en) * | 2019-01-15 | 2020-07-16 | Tangen Bioscience Inc. | Method for suppressing non-specific amplification products in nucleic acid amplification technologies |
CN111926119B (zh) * | 2020-09-03 | 2023-04-21 | 上海市计量测试技术研究院 | 一种用于检测新型冠状病毒的核酸检测试剂盒及其使用方法 |
GB202018125D0 (en) | 2020-11-18 | 2020-12-30 | Aidian Oy | Method |
NL2033124B1 (en) | 2022-09-23 | 2024-03-29 | Rapidemic B V | Methods and device for multiple-label nucleic acid amplification and detection |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4800159A (en) | 1986-02-07 | 1989-01-24 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences |
EP1073766A1 (en) * | 1998-04-29 | 2001-02-07 | Trustees Of Boston University | Methods and compositions pertaining to pd-loops |
US6432642B1 (en) * | 1999-01-15 | 2002-08-13 | Pe Corporation (Ny) | Binary probe and clamp composition and methods for a target hybridization detection |
CA2365668C (en) | 1999-03-17 | 2014-05-20 | The Board Of Trustees Of The Leland Stanford Junior University | In vitro macromolecule biosynthesis methods using exogenous amino acids and a novel atp regeneration system |
CA2382462A1 (en) * | 1999-08-20 | 2001-03-01 | Roche Diagnostics Gmbh | Enzymatic synthesis of deoxyribonucleosides |
EP1218542B1 (en) * | 1999-09-13 | 2004-03-24 | Nugen Technologies, Inc. | Methods and compositions for linear isothermal amplification of polynucleotide sequences |
US7399590B2 (en) * | 2002-02-21 | 2008-07-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
EP1759012B1 (en) * | 2004-06-01 | 2013-05-22 | Alere San Diego, Inc. | Recombinase polymerase amplification |
WO2006051988A1 (ja) * | 2004-11-15 | 2006-05-18 | Riken | 核酸の増幅方法 |
EP1866434B1 (en) | 2005-02-19 | 2013-06-05 | Avacta Group plc | Isothermal nucleic acid amplification |
JP2006271372A (ja) | 2005-03-01 | 2006-10-12 | Yamasa Shoyu Co Ltd | 糖鎖の製造法 |
EP1929049B1 (en) * | 2005-07-25 | 2013-04-10 | Alere San Diego, Inc. | Methods for multiplexing recombinase polymerase amplification |
US8071308B2 (en) * | 2006-05-04 | 2011-12-06 | Alere San Diego, Inc. | Recombinase polymerase amplification |
EP3249056B1 (en) | 2008-06-11 | 2019-04-10 | GeneForm Technologies Limited | Method of atp regeneration in a recombinase reaction |
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2009
- 2009-06-11 EP EP17179338.3A patent/EP3249056B1/en active Active
- 2009-06-11 LT LTEP09762016.5T patent/LT2304054T/lt unknown
- 2009-06-11 SI SI200931719T patent/SI2660336T1/sl unknown
- 2009-06-11 HU HUE13178545A patent/HUE034561T2/en unknown
- 2009-06-11 PT PT97620165T patent/PT2304054T/pt unknown
- 2009-06-11 PT PT131785453T patent/PT2660336T/pt unknown
- 2009-06-11 PL PL09762016T patent/PL2304054T3/pl unknown
- 2009-06-11 JP JP2011513055A patent/JP5798917B2/ja active Active
- 2009-06-11 ES ES09762016.5T patent/ES2638853T3/es active Active
- 2009-06-11 WO PCT/GB2009/050662 patent/WO2009150467A1/en active Application Filing
- 2009-06-11 EP EP09762016.5A patent/EP2304054B1/en active Active
- 2009-06-11 US US12/997,382 patent/US9062344B2/en active Active
- 2009-06-11 HU HUE09762016A patent/HUE036005T2/hu unknown
- 2009-06-11 EP EP13178545.3A patent/EP2660336B1/en active Active
- 2009-06-11 DK DK13178545.3T patent/DK2660336T3/en active
- 2009-06-11 PL PL13178545T patent/PL2660336T3/pl unknown
- 2009-06-11 LT LTEP13178545.3T patent/LT2660336T/lt unknown
- 2009-06-11 CN CN201510103597.7A patent/CN104862385B/zh active Active
- 2009-06-11 DK DK09762016.5T patent/DK2304054T3/en active
- 2009-06-11 CN CN200980131223.9A patent/CN102119225B/zh active Active
- 2009-06-11 SI SI200931718T patent/SI2304054T1/sl unknown
- 2009-06-11 CA CA2727212A patent/CA2727212C/en active Active
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2010
- 2010-12-06 ZA ZA2010/08749A patent/ZA201008749B/en unknown
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2015
- 2015-05-12 US US14/709,793 patent/US9657340B2/en active Active
- 2015-08-21 JP JP2015164059A patent/JP6301881B2/ja active Active
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2017
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- 2017-08-28 HR HRP20171286TT patent/HRP20171286T1/hr unknown
- 2017-08-31 CY CY20171100921T patent/CY1119346T1/el unknown
- 2017-09-08 HR HRP20171356TT patent/HRP20171356T1/hr unknown
- 2017-09-11 CY CY20171100958T patent/CY1119331T1/el unknown
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