ES2578103T3 - Mutantes de ADN polimerasa de phi29 que tienen mayor termoestabilidad y capacidad de procesamiento que comprenden M8R, V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, y F526L - Google Patents
Mutantes de ADN polimerasa de phi29 que tienen mayor termoestabilidad y capacidad de procesamiento que comprenden M8R, V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, y F526L Download PDFInfo
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- 102000016928 DNA-directed DNA polymerase Human genes 0.000 title abstract description 12
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 title abstract description 12
- 102220586964 Molybdopterin synthase sulfur carrier subunit_V51A_mutation Human genes 0.000 title abstract description 3
- 102220172075 rs150884181 Human genes 0.000 title abstract description 3
- 102220012914 rs397516364 Human genes 0.000 title abstract description 3
- 230000035772 mutation Effects 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 241000701844 Bacillus virus phi29 Species 0.000 abstract 3
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 7
- 239000000758 substrate Substances 0.000 description 4
- 230000004568 DNA-binding Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020001019 DNA Primers Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000013412 genome amplification Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- 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
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- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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Abstract
Una ADN polimerasa del bacteriófago phi29 que comprende una mutación M8R, en donde la ADN polimerasa del bacteriófago phi29 tiene mayor estabilidad de proteínas en comparación con la ADN polimerasa de phi29 de tipo salvaje, y, opcionalmente, en donde la ADN polimerasa del bacteriófago phi29 comprende adicionalmente al menos una mutación seleccionada de V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, y F526L.
Description
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Es muy probable que la enzima de tipo salvaje que lleva a cabo la reacción de WGA a 30°C tienda a amplificar las regiones del genoma con un menor contenido de CG. El sesgo de la amplificación de CG podría ser reducido significativamente realizando WGA con las polimerasas Mut_4 y Mut_5 a 37°C y 42°C respectivamente. La reducción del sesgo de amplificación también debe dar lugar a disminución del efecto de la pérdida de alelo (ADO).
Ejemplo 5
Análisis de unión a ADN demutantes de polimerasa de phi29
El análisis de desplazamiento de la movilidad electroforética (EMSA) se realizó utilizando como sustrato la molécula de ADN de 15 unidades/21 unidades híbrida marcada para determinar si nuevas mutaciones de la ADN polimerasa de phi29 potenciaron la unión de la enzima al ADN. El oligonucleótido de 15 unidades (5'-GATCACAGTGAGTAC-3') (SEQ ID NO: 8) se marcó en 5' con ATP [γ-32P]utilizando polinucleótido quinasa de T4 y se hibridó con el oligonucleótido de 21 unidades (5'-TCTATTGTACTCACTGTGATC-3') (SEQ ID NO: 9) en presencia de NaCl 0,2 M y Tris-HCl 60 mM, pH 7,5 (De Vega, M. et al., 2010). La molécula híbrida de 15 unidades/21 unidades marcado en 5' resultante se utilizó como cebador/molde de ADN para analizar la interacción con las enzimas de phi29 de tipo salvaje o mutantes que contenían mutaciones únicas o múltiples. La mezcla de incubación contenía, en un volumen final de 20 µl, Tris-acetato 33 mM de pH 7,9, acetato de potasio 66 mM, glicerol al 10%, 0,1 mg/ml de BSA, 2 pM del sustrato de ADN de 15 unidades/21 unidades/ADN, y cantidades crecientes de la enzima correspondiente (0,10, 20, 40, 80, 160, 300, 600 pM). Después de incubación durante cinco minutos a 30°C, las muestras se sometieron a electroforesis en geles de poliacrilamida al 10% (p/v) (29:1, acrilamida: bisacrilamida) que contenían Tris-acetato 40 mM de pH 8,4, EDTA 1 mM (tampón 1XTAE). La electroforesis se realizó en el mismo tampón 1XTAE a temperatura ambiente durante dos horas a aproximadamente 8-9 V/cm. Los geles de EMSA fueron analizados mediante Soporte Lógico de Análisis de Imágenes Typhoon Trio y OptiQuant™. Los valores de Kd del complejo enzima-oligonucleótido se calcularon mediante soporte lógico GraphPad Prism versión 4.03 utilizando la ecuación: [E-ADN] = ([DNAo] + [Eo]
+ Kd -(([DNAo] + [Eo] + Kd)2 -4 [DNAo] [Eo]0.5)/2; donde [DNA-E] -es la concentración del complejo enzimaoligonucleótido, [DNAo] -Concentración total de oligonucleótidos (2 pM), [Eo] -concentracióntotal de enzima.
La FIG. La figura 8 muestra un EMSA representativo de la unión de enzima wt, Mu_4 y Mut_5 a ADN. La molécula híbrida marcada en 5' de 15 unidades/21 unidades (ADNdh) se incubó con ADN polimerasa de phi29 o con la ADN polimerasa mutante. Después de la electroforesis en gel, la movilidad de ADNdh libre y del complejo de la polimerasa-ADN se detectó mediante autorradiografía. La FIG. 8 es representativa de diversos experimentos (experimentos con mutantes que contienen mutaciones individuales no mostrados). Todos los valores numéricos de la constante de disociación determinadamediante análisis EMSA se resumen en la Tabla 5.
Tabla 5. Constantes de disociación(Kd) de los mutantes de la ADN polimerasa de phi29
Las enzimas mutantes mostraron una mejor unión al ADN (menor Kd), requiriendo una concentración de enzima aproximadamente dos veces o tres veces menor en comparación con la polimerasa de tipo salvaje para generar una cantidad similar de complejo de proteína y ADN. Los valores de la constante de disociación (Kd) de algunos mutantes que contenían mutaciones individuales (M8R, V51A, M97T), así como de la polimerasa Mut_5 fueron más bajos que los de la ADN polimerasa de phi29 de tipo salvaje (Tabla 5). De Vega et al. 2010 mostraron que la ADN polimerasa de phi29 con dominio de unión a ADN adicional tenían mayor afinidad por el sustrato, y posteriormente también se mejoraron otras características de enzimas como la capacidad de procesamiento y el rendimiento de amplificación. Puesto que los mutantes de ADN polimerasa de phi29 descritos también poseen una mayor afinidad por el sustrato, sería razonableesperar quetales mutantes muestren mejoras en otras características importantes de esta enzima.
Referencias:
- 1.
- Akeson et al. (2010) Processive Replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J. Am. Chem. Soc. Vol. 132 Núm. 50 págs. 17961-17972
- 2.
- Alsmadi et al. (2009) Specific and complete human genome amplification with improved yield achieved by 15
Claims (1)
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imagen1
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361815893P | 2013-04-25 | 2013-04-25 | |
| US201361815893P | 2013-04-25 | ||
| US14/135,860 US9422535B2 (en) | 2013-04-25 | 2013-12-20 | phi29 DNA polymerase mutants having increased thermostability and processivity |
| US201314135860 | 2013-12-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2578103T3 true ES2578103T3 (es) | 2016-07-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES14165903.7T Active ES2578103T3 (es) | 2013-04-25 | 2014-04-24 | Mutantes de ADN polimerasa de phi29 que tienen mayor termoestabilidad y capacidad de procesamiento que comprenden M8R, V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, y F526L |
Country Status (3)
| Country | Link |
|---|---|
| US (2) | US9422535B2 (es) |
| EP (2) | EP2813576B1 (es) |
| ES (1) | ES2578103T3 (es) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017109262A1 (es) | 2015-12-24 | 2017-06-29 | Consejo Superior De Investigaciones Científicas (Csic) | VARIANTES DE LA ADN POLIMERASA DEL BACTERIÓFAGO phi29 CON TERMOACTIVIDAD MEJORADA |
| CN110892067B (zh) * | 2017-04-27 | 2023-05-02 | 深圳华大智造科技股份有限公司 | 热稳定性提高的phi29 DNA聚合酶突变体 |
| CN110719955B (zh) * | 2017-07-28 | 2023-05-02 | 深圳华大智造科技股份有限公司 | 提高了热稳定性的Phi29 DNA聚合酶突变体及其应用 |
| EP3728633A1 (en) | 2017-12-22 | 2020-10-28 | Thermo Fisher Scientific Baltics Uab | Polymerase chain reaction composition comprising amines |
| CN108588202B (zh) * | 2018-05-14 | 2019-06-04 | 南京诺唯赞生物科技有限公司 | 一种单细胞全基因扩增方法 |
| US10934533B1 (en) | 2018-10-10 | 2021-03-02 | New England Biolabs, Inc. | Variant DNA polymerases having improved properties and method for improved isothermal amplification of a target DNA |
| JP7256280B2 (ja) * | 2018-10-11 | 2023-04-11 | 深▲せん▼華大生命科学研究院 | 熱安定性が向上したＰｈｉ２９ ＤＮＡポリメラーゼ突然変異体及びそのシーケンシングにおける応用 |
| ES3015001T3 (en) * | 2019-05-17 | 2025-04-28 | 4Basebio Uk Ltd | Phi29 dna polymerase mutants with improved primer recognition |
| CA3170318A1 (en) * | 2020-02-10 | 2021-08-19 | BioSkryb Genomics, Inc. | Phi29 mutants and use thereof |
| CN115698275A (zh) * | 2020-06-10 | 2023-02-03 | 深圳华大生命科学研究院 | 稳定且酶活性高的Phi29 DNA聚合酶及其编码基因与应用 |
| WO2022015600A2 (en) | 2020-07-13 | 2022-01-20 | Singular Genomics Systems, Inc. | Methods of sequencing complementary polynucleotides |
| EP4208470A4 (en) | 2020-10-22 | 2024-10-30 | Singular Genomics Systems, Inc. | NUCLEIC ACID CIRCULARIZATION AND AMPLIFICATION ON A SURFACE |
| US11486001B2 (en) | 2021-02-08 | 2022-11-01 | Singular Genomics Systems, Inc. | Methods and compositions for sequencing complementary polynucleotides |
| WO2024076991A2 (en) | 2022-10-03 | 2024-04-11 | Singular Genomics Systems, Inc. | Modified enzymes and uses thereof |
| GB202310278D0 (en) | 2023-07-05 | 2023-08-16 | Navinci Diagnostics Ab | Stabilisation of phi29 polymerase |
| WO2025202929A1 (en) | 2024-03-29 | 2025-10-02 | Pfizer Inc. | Methods for producing nucleic acids |
| US20250313876A1 (en) | 2024-04-06 | 2025-10-09 | Ribbon Bio GmbH | Isolation of dna fragments |
| CN119082075B (zh) * | 2024-09-12 | 2025-06-27 | 湖南艾科瑞生物工程有限公司 | 性能提升的phi29 DNA聚合酶突变体及其制备方法与应用 |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5198543A (en) | 1989-03-24 | 1993-03-30 | Consejo Superior Investigaciones Cientificas | PHI29 DNA polymerase |
| US5196543A (en) | 1989-10-17 | 1993-03-23 | Delalande S.A. | 3-aryloxazolidinone derivatives, process for their preparation and their use in therapy |
| ES2648313T3 (es) | 2005-12-22 | 2017-12-29 | Pacific Biosciences Of California, Inc. | Polimerasas para la incorporación de análogos nucleotídicos |
| US20100009355A1 (en) | 2006-03-14 | 2010-01-14 | Kolodney Michael S | Selective amplification of minority mutations using primer blocking high-affinity oligonucleotides |
| WO2008051530A2 (en) | 2006-10-23 | 2008-05-02 | Pacific Biosciences Of California, Inc. | Polymerase enzymes and reagents for enhanced nucleic acid sequencing |
| WO2009091847A2 (en) | 2008-01-14 | 2009-07-23 | Life Technologies Corporation | Compositions, methods and systems for single molecule sequencing |
| ES2614078T3 (es) | 2008-03-31 | 2017-05-29 | Pacific Biosciences Of California, Inc. | Generación de polimerasas modificadas para precisión mejorada en secuenciación de una única molécula |
| US9127259B2 (en) | 2008-03-31 | 2015-09-08 | Pacific Biosciences Of California, Inc. | Enzymes resistant to photodamage |
| US8999676B2 (en) * | 2008-03-31 | 2015-04-07 | Pacific Biosciences Of California, Inc. | Recombinant polymerases for improved single molecule sequencing |
| US8420366B2 (en) | 2008-03-31 | 2013-04-16 | Pacific Biosciences Of California, Inc. | Generation of modified polymerases for improved accuracy in single molecule sequencing |
| 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 |
| US8906660B2 (en) | 2012-02-01 | 2014-12-09 | Pacific Biosciences Of California, Inc. | Recombinant polymerases with increased phototolerance |
| US20140094374A1 (en) * | 2012-10-01 | 2014-04-03 | Pacific Biosciences Of California, Inc. | Recombinant Polymerases with Increased Readlength and Stability for Single-Molecule Sequencing |
-
2013
- 2013-12-20 US US14/135,860 patent/US9422535B2/en active Active
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| Publication number | Publication date |
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| EP2813576B1 (en) | 2016-06-15 |
| EP3093347B1 (en) | 2019-08-07 |
| US20170015980A1 (en) | 2017-01-19 |
| US9885025B2 (en) | 2018-02-06 |
| EP3093347A1 (en) | 2016-11-16 |
| EP2813576A3 (en) | 2015-03-18 |
| EP2813576A2 (en) | 2014-12-17 |
| US9422535B2 (en) | 2016-08-23 |
| US20140322759A1 (en) | 2014-10-30 |
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