ES2632768T3 - Flujo de trabajo para la detección de ligandos empleando ácidos nucleicos - Google Patents
Flujo de trabajo para la detección de ligandos empleando ácidos nucleicos Download PDFInfo
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- ES2632768T3 ES2632768T3 ES12702099.8T ES12702099T ES2632768T3 ES 2632768 T3 ES2632768 T3 ES 2632768T3 ES 12702099 T ES12702099 T ES 12702099T ES 2632768 T3 ES2632768 T3 ES 2632768T3
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Classifications
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- 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
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
-
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
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- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
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- General Engineering & Computer Science (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Un método para acoplar al menos dos oligonucleótidos para producir un oligonucleótido acoplado y amplificar el oligonucleótido acoplado, en el que el acoplamiento y la amplificación se producen en una única mezcla de reacción, comprendiendo dicho método las etapas, en combinación, de: a) poner en contacto una proteína diana o analito con al menos una primera y una segunda sonda, teniendo cada sonda una especificidad de unión por la proteína o el analito y estando unida al menos a un tipo de oligonucleótido; b) acoplar entre sí los oligonucleótidos sobre la primera y la segunda sonda empleando (i) una ligasa seleccionada del grupo que consiste en la ligasa de SEQ ID NO:77, la ligasa de SEQ ID NO:78, la ligasa de SEQ ID NO:79, la ligasa de SEQ ID NO:80, la ligasa de SEQ ID NO:81, la ligasa de SEQ ID NO:82, y sus combinaciones, y (ii) un puente oligonucleotídico, en el que dicho puente oligonucleotídico comprende un extremo 3' de cuatro a nueve bases de longitud, y un extremo 5' de cuatro a nueve bases de longitud, para producir un ácido nucleico diana y amplificar el ácido nucleico diana; y, c) detectar el ácido nucleico diana amplificado
Description
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15
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55
5.476.930; Fung et al., patente de EE. UU. n.º 5.593.826; Kool, patente de EE. UU. n.º 5.426.180; Landegren et al., patente de EE. UU. n.º 5.871.921; Xu y Kool, Nucleic Acids Research, 27:875-881 (1999); Higgins et al., Methods in Enzymology, 68:50-71 (1979); Engler et al., The Enzymes, 15-3-29 (1982); y Namsaraev, publicación de patente de EE. UU. 2004/0110213. Se ha indicado la fidelidad de varias ligasas conocidas, basándose, por ejemplo, en la evaluación de las tasas de acoplamiento o de acoplamiento desapareado. Por ejemplo, se ha indicado que la ligasa dependiente de NAD+ de la bacteria hipertermófila Aquifex aeolicus genera productos de acoplamiento erróneo 3' detectables con desapareamientos de C:A, T:G, y G:T (Tong et al., Nucl. Acids Res., 28(6):1447-1454, 2000); se ha indicado que una preparación parcialmente purificada de la ADN ligasa III bovina genera productos de acoplamiento erróneo 3' detectables con desapareamientos de C:T, G:T, y T:G, mientras que la ligasa I humana genera productos de acoplamiento erróneo 3' detectables con desapareamientos de C:T y G:T, pero no de T:G (Husain et al., J. Biol. Chem., 270(16):9683-9690, 1995); y se ha indicado que la ADN ligasa de la bacteria termófila Thermus thermophilus (Tth) genera niveles detectables de productos de acoplamiento erróneo 3' detectables con desapareamientos de T:G y G:T (Luo et al., Nucl. Acids Res., 24(14):3071-3078, 1996). La ADN ligasa del bacteriófago T4 genera productos de acoplamiento erróneo detectables con una amplia gama de sustratos desapareados y parece tener menor fidelidad que las ligasas de especies de Thermus en al menos uno a dos órdenes de magnitud (Landegren et al., Science, 241:1077-1080, 1988; Tong et al., Nucl. Acids Res., 27(3):788-794, 1999).
Un ensayo particularmente útil es el ensayo de acoplamiento de oligonucleótidos (OLA). El OLA es un método conveniente y altamente riguroso que permite la distinción entre variantes de secuencias de ADN conocidas (Landegren, 1988). Por ejemplo, el análisis múltiplex de loci altamente polimorfos es útil para la identificación de individuos, por ejemplo, para ensayos de paternidad y en la ciencia forense, la correspondencia entre donantes y receptores en transplantes de órganos, el diagnóstico de enfermedades genéticas, la prognosis, y el asesoramiento prenatal y otros ensayos con base genética que dependen de la discriminación de diferencias de una sola base en múltiples loci (Delahunty, 1996). Los productos de un OLA múltiplex pueden separarse de modo electroforético y de las sondas no acopladas bajo condiciones desnaturalizantes con detección de fluorescencia (Grossman, 1994). Por ejemplo, dos quimeras de PNA-ADN, una quimera de secuencia de tipo salvaje ("wild-type", WT) y una quimera de secuencia mutante pueden llevar diferentes tintes fluorescentes. Solo cuando la secuencia mutante está presente en la muestra diana es cuando la quimera de secuencia mutante se acopla a la segunda sonda (oligo) acoplada en posición adyacente si el par de bases mutante está en el sitio de acoplamiento. Los productos del acoplamiento pueden separarse basándose en: (i) el tamaño, empleando electroforesis y/o cromatografía y/o (ii) marcadores detectables (Grossman, 1994). Con una pluralidad de tintes fluorescentes como marcadores de quimeras con secuencias que se dirigen a secuencias diana exclusivas, pueden realizarse OLA múltiplex en una sola muestra en un único recipiente. Los requisitos para un OLA múltiplex eficaz incluyen sondas que se asocian y se acoplan de una manera muy específica y rápida. Las quimeras y las secuencias de la segunda sonda pueden seleccionarse de modo que la base mutante, o polimorfismo de una sola base, pueda estar en el 5'-fosfato de la segunda sonda o en el 3'-terminal de la quimera. Se contempla que los experimentos de OLA de la presente invención puedan realizarse sobre soportes sólidos, en los que el ácido nucleico molde, la sonda quimérica de PNA-ADN, o la segunda sonda pueden estar inmovilizados sobre una partícula sólida o esfera, o una superficie sólida porosa o no porosa. Cuando están inmovilizados, el molde, la quimera o la segunda sonda preferiblemente están unidos covalentemente al sustrato sólido, por ejemplo, a través de una unidad de monómero terminal. El sustrato sólido puede ser poliestireno, vidrio de tamaño de poro controlado, gel de sílice, sílice, poliacrilamida, esferas magnéticas, hidroxietilmetacrilato, poliamida, polietileno, polietileoxi, y copolímeros e injertos de cualquiera de los anteriores sustratos sólidos. La configuración o el formato del sustrato sólido puede ser de pequeñas partículas o esferas con un diámetro de aproximadamente 1 a 50 µm, membranas, fritas, portaobjetos, placas, chips fabricados con micromáquinas, capas de alcantiol-oro, superficies no porosas y medios inmovilizantes de polinucleótidos.
Tal como se describió anteriormente, el acoplamiento enzimático generalmente se logra empleando una ligasa, que puede ser un polipéptido. Las ligasas adecuadas incluyen, por ejemplo, ácido nucleico ligasa, oligonucleótido ligasa, ADN ligasa, ARN ligasa, y similares. Las ARN ligasas adecuadas incluyen las descritas o empleadas, por ejemplo, en cualquiera de las patentes de EE. UU. n.os 4.582.802; 5.665.545; 6.194.637; 6.444.429; 6.455.274; 6.576.453; 6.635.425; 6.855.523; 7.811.753; y/o cualquiera de las publicaciones de patente de EE. UU. 2004/0171047A1, 2004/0191871A1, US20050266487A1, 2006/0223098A1, 2007/0037190A1, 20080160526A1; 2009/0061481A1, 2010/0099683A1, y/o 2010/0184618A1. Los ejemplos de ADN ligasas pueden incluir, por ejemplo ADN ligasa de T3, ADN ligasa de T4, ADN ligasa de T5, ADN ligasa de T7, ADN ligasa del virus de vaccinia, ADN ligasa de E. coli, ADN ligasa I de mamífero, ADN ligasa II de mamífero, ADN ligasa III de mamífero, ADN ligasa de Tth, ADN ligasa de KOD, una ADN ligasa termoestable y/o sus derivados, fragmentos y/o combinaciones. Las ARN ligasas adecuadas incluyen las descritas o empleadas, por ejemplo, en cualquiera de las patentes de EE. UU. n. os 4.661.450; 5.516.664; 5.602.000; 5.807.674; 6.368.801; 6.492.161; 6.635.453; o cualquiera de las publicaciones de patente de EE. UU. n. os 2003/0082536A1; 2004/0058330A1; 2005/0266439A1; 2005/0074774A1; 2008/0045418A1; 2010/00159526A1. Los ejemplos de ARN ligasas pueden incluir, por ejemplo, ARN ligasa de T4, ARN ligasa del bacteriófago RB69, ARN ligasa del virus de la polihedrosis nuclear Autographa californica, una ARN ligasa termófila, ARN ligasa del bacteriófago RM378, ARN ligasa del bacteriófago TS2126 y/o sus derivados, fragmentos y/o combinaciones.
En algunas realizaciones, la ligasa es una "ligasa de huella pequeña" ("small footprint ligase", SFL). Una SFL tiene la capacidad de acoplarse a polinucleótidos cortos (por ejemplo, al menos aproximadamente 3 nucleótidos). Tal como
12
fragmentos funcionales o variantes. Los ejemplos representativos de SFL incluyen ligasa CV, DLX, DLXd, DLXd2 y ligasa MnM. Una SFL preferida es la ligasa del virus de Chlorella. Se identifican algunos ejemplos de ligasas y se indica su GI o número de registro en la siguiente Tabla 1:
Tabla 1
- PRK08224
-
imagen12 imagen13
- B. Acidobacteria
-
imagen14 imagen15
- Bacteria; grupo de Fibrobacteres/Acidobacteria; Acidobacteria; Acidobacteria no clasificada; Candidatus Koribacter; Candidatus Koribacter versatilis
-
imagen16 imagen17
- Candidatus Solibacter usitatus Ellin6076Candidatus Solibacter (1 proteína)
- ADN ligasa dependiente de ATP YP_826317
- imagen18
-
imagen19 imagen20
- C. Actinobacteria
-
imagen21 imagen22
- Bacteria; Actinobacteria; Actinobacteria (clase); Actinobacteridae; Actinomycetales; Corynebacterineae; Mycobacteriaceae; Mycobacterium; Mycobacterium marinum
-
imagen23 imagen24
- Mycobacterium gilvum PYR-GCKMycobacterium (26 proteínas)
- ADN ligasa dependiente de ATP YP_001132524
- Mycobacterium vanbaalenii PYR-1Mycobacterium (26 proteínas)
- ADN ligasa dependiente de ATP YP_956315
- Mycobacterium sp. MCSMycobacterium (26 proteínas)
- ADN ligasa dependiente de ATP YP_642076
- imagen25
-
imagen26 imagen27
- F. Chlamydiae/Verrucomicrobia
-
imagen28 imagen29
- Bacteria; grupo de Chlamydiae/Verrucomicrobia; Verrucomicrobia; Opitutae; Opitutales; Opitutaceae; Opitutus; Opitutus terrae
-
imagen30 imagen31
- Opitutus terrae PB90-10pitutus (1 proteína)
- ADN ligasa dependiente de ATP YP_001821013
- imagen32
-
imagen33 imagen34
- PRK09125
-
imagen35 imagen36
- Organismo
- Nombre de la proteína n.º de registro
- O. Betaproteobacteria
-
imagen37 imagen38
- Neisseria meningitidis Z2491Neisseria (7 proteínas)
- ADN ligasa YP_002341892
- Thiobacillus denitrificans ATCC 25259Thiobacillus (1 proteínas)
- ADN ligasa YP_314570
- Variovorax paradoxus S110Variovorax (1 proteínas)
- ADN ligasa YP_002944627
14
- PRK08224
-
imagen39 imagen40
- B. Acidobacteria
-
imagen41 imagen42
- Verminephrobacter eiseniae EF01-2Verminephrobacter (1 proteínas)
- ADN ligasa YP_998235
- imagen43
-
imagen44 imagen45
- P. Deltaproteobacteria
- - -
- Desulfobacterium autotrophicum HRM2Desulfobacterium (1 proteína)
- LigA2 YP_002604477
- Myxococcus xanthus DK 1622Myxococcus (1 proteína)
- ADN ligasa YP_628883
- imagen46
- - -
- Q. Epsilonproteobacteria
-
imagen47 imagen48
- Campylobacter jejuni subsp. jejuni NCTC 11168Campylobacter (10 proteínas)
- ADN ligasa dependiente de ATP YP_002345037
- Sulfurimonas denitrificans DSM 1251Sulfurimonas (1 proteínas)
- ADN ligasa YP_393098
- imagen49
- - -
- R. Gammaproteobacteria
-
imagen50 imagen51
- Aggregatibacter aphrophilus NJ8700Aggregatibacter (2 proteínas)
- ADN ligasa dependiente de ATP YP_003007537
- Haemophilus influenzae PittEEHaemophilus (3 proteínas)
- ADN ligasa dependiente de ATP YP_001290961
- Shewanella baltica OS195Shewanella (18 proteínas)
- ADN ligasa dependiente de ATP YP_001554317
- Shewanella loihica PV-4Shewanella (18 proteínas)
- ADN ligasa dependiente de ATP YP_001093713
- Vibrio cholerae M66-2Vibrio (9 proteínas)
- ADN ligasa YP_002810248
- imagen52
-
imagen53 imagen54
- PHA0454
-
imagen55 imagen56
- Organismo
- Nombre de la proteína n.º de registro
- b. Virus
- - -
- Virus; virus de ADNbc, sin etapa de ARN; Caudovirales; Podoviridae; Autographivirinae; virus similares a phiKMV
-
imagen57
- virus similares al fago de LKD16phiKMV de Pseudomonas (7 proteínas)
- ADN ligasa dependiente de ATP YP_001522807
15
- PRK08224
-
imagen58 imagen59
- B. Acidobacteria
-
imagen60 imagen61
- imagen62
-
imagen63 imagen64
- CLSZ2445448
-
imagen65 imagen66
- Organismo
- Nombre de la proteína n.º de registro
- a. Eukaryota
-
imagen67 imagen68
- Eukaryota; Alveolata; Ciliophora; Intramacronucleata; Oligohymenophorea; Peniculida; Parameciidae; Paramecium; Paramecium tetraurelia
-
imagen69 imagen70
- Paramecium tetraurelia strain d4-2Paramecium (5 proteínas)
- ADN ligasa XP_001347270
- imagen71
-
imagen72 imagen73
- PRK07636
-
imagen74 imagen75
- Organismo
- Nombre de la proteína n.º de registro
- J. Firmicutes
-
imagen76 imagen77
- Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus clausii
-
imagen78 imagen79
- Bacillus subtilis subsp. subtilis str. 168Bacillus
- ADN ligasa dependiente de ATP NP_389932
- Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Geobacillus
-
imagen80 imagen81
- Geobacillus sp. Y412MC10Geobacillus
- ADN ligasa dependiente de ATP YP_003240778
- CLSK2551528
-
imagen82 imagen83
- Organismo
- Nombre de la proteína n.º de registro
- J. Firmicutes
-
imagen84 imagen85
- Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Geobacillus
-
imagen86 imagen87
- Geobacillus sp. Y412MC10Geobacillus (1 proteína)
- ADN ligasa dependiente de ATP YP_003245332
- imagen88
- - -
- CLSK2470953
-
imagen89 imagen90
- Organismo
- Nombre de la proteína n.º de registro
- C. Actinobacteria
-
imagen91 imagen92
16
- Bacteria; Actinobacteria; Actinobacteria (clase); Actinobacteridae; Actinomycetales; Micrococcineae; Micrococcaceae; Arthrobacter; Arthrobacter chlorophenolicus
-
imagen93
- Arthrobacter chlorophenolicus A6 (plasmid)Arthrobacter (2 proteínas)
- ADN ligasa dependiente de ATP YP_002478427
- imagen94
-
imagen95 imagen96
- CLSK2469924
-
imagen97 imagen98
- Organismo
- Nombre de la proteína n.º de registro
- J. Firmicutes
-
imagen99 imagen100
- Bacteria; Firmicutes; Bacilli; Bacillales; Alicyclobacillaceae; Alicyclobacillus; Alicyclobacillus acidocaldarius; Alicyclobacillus acidocaldarius subsp. acidocaldarius
-
imagen101 imagen102
- Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446Alicyclobacillus
- ADN ligasa dependiente de ATP YP_003185050
- imagen103
-
imagen104 imagen105
- CLSK2340991
-
imagen106 imagen107
- Organismo
- Nombre de la proteína n.º de registro
- N. Alphaproteobacteria
-
imagen108 imagen109
- Bacteria; Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Phenylobacterium; Phenylobacterium zucineum
- Phenylobacterium zucineum HLK1 (plásmido)Phenylobacterium (2 proteínas)
- ADN ligasa dependiente de ATP YP_002128631
- imagen110
- - -
- CLSK2333706
-
imagen111 imagen112
- Organismo
- Nombre de la proteína n.º de registro
- J. Firmicutes
-
imagen113 imagen114
- Bacteria; Firmicutes; Clostridia; Clostridiales; Peptococcaceae; Candidatus Desulforudis; Candidatus Desulforudis audaxviator
- Candidatus Desulforudis audaxviator MP104CCandidatus Desulforudis (1 proteína)
- ADN ligasa dependiente de ATP YP_001716762
- imagen115
- - -
- CLSK962101
-
imagen116 imagen117
- Organismo
- Nombre de la proteína n.º de registro
17
- C .Actinobacteria
-
imagen118 imagen119
- Bacteria; Actinobacteria; Actinobacteria (clase); Actinobacteridae; Actinomycetales; Micromonosporineae; Micromonosporaceae; Salinispora; Salinispora arenicola
- - -
- Salinispora arenicola CNS-205Salinispora (2 proteínas)
- región de la ligasa LigD de ADN polimerasa YP_001539124
- Salinispora tropica CNB-440Salinispora (2 proteínas)
- ADN ligasa dependiente de ATP YP_001160776
- imagen120
- - -
- CLSK915249
-
imagen121 imagen122
- Organismo
- Nombre de la proteína n.º de registro
- C. Actinobacteria (véase CLSK2303611 anterior)
-
imagen123 imagen124
- Bacteria; Actinobacteria; Actinobacteria (clase); Actinobacteridae; Actinomycetales; Streptomycineae; Streptomycetaceae; Streptomyces; Streptomyces coelicolor
-
imagen125 imagen126
- Streptomyces avermitilis MA-4680 (plásmido)Streptomyces (2 proteínas)
- ADN ligasa dependiente de ATP putativa NP_828839
- Streptomyces sp. HK1 (plásmido)Streptomyces (2 proteínas)
- ADN ligasa dependiente de ATP putativa YP_001661618
- CLSK862724
-
imagen127 imagen128
- Organismo
- Nombre de la proteína n.º de registro
- A. Archaea
-
imagen129 imagen130
- Archaea; Euryarchaeota; Archaeoglobi; Archaeoglobales; Archaeoglobaceae; Archaeoglobus; Archaeoglobus fulgidus
-
imagen131 imagen132
- Archaeoglobus fulgidus DSM 4304Archaeoglobus (1 proteína)
- ADN ligasa, putativa NP_070553
- imagen133
-
imagen134 imagen135
- J. Firmicutes
-
imagen136 imagen137
- Pelotomaculum thermopropionicum SIPelotomaculum (1 proteína)
- ADN ligasa dependiente de ATP YP_001211793
- Thermoanaerobacter pseudethanolicus ATCC 33223Thermoanaerobacter (2 proteínas)
- ADN ligasa dependiente de ATP YP_001664477
- imagen138
-
imagen139 imagen140
- CLSK820690
-
imagen141 imagen142
- Organismo
- Nombre de la proteína n.º de registro
18
- A. Archaea
-
imagen143 imagen144
- Archaea; Euryarchaeota; muestras ambientales
-
imagen145 imagen146
- muestras ambienteles RC-I de arqueas metanogénicas no cultivadas (1 proteína)
- ADN ligasa dependiente de ATP YP_686457
- N. Alphaproteobacteria
-
imagen147 imagen148
- Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Bradyrhizobiaceae; Bradyrhizobium; Bradyrhizobium japonicum
-
imagen149 imagen150
- Bradyrhizobium japonicum USDA 110Bradyrhizobium (2 proteínas)
- ADN ligasa NP_774671
- Bradyrhizobium sp. BTAilBradyrhizobium (2 proteínas)
- ADN ligasa dependiente de ATP putativa YP_001243518
- imagen151
-
imagen152 imagen153
- CLSK808255
-
imagen154 imagen155
- Organismo
- Nombre de la proteína n.º de registro
- N. Alphaproteobacteria
-
imagen156 imagen157
- Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Rhizobiaceae; grupo de SinorhizobiumlEnsifer; Sinorhizobium; Sinorhizobium medicae
-
imagen158 imagen159
- Sinorhizobium medicae WSM419Sinorhizobium (2 proteínas)
- región de la ligasa LigD de ADN polimerasa YP_001326990
- Sinorhizobium meliloti 1021 (plasmid)Sinorhizobium (2 proteínas)
- proteína de ADN ligasa dependiente de ATP putativa NP_437750
- CLSK806855
-
imagen160 imagen161
- Organismo
- Nombre de la proteína n.º de registro
- N. Alphaproteobacteria
-
imagen162 imagen163
- Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Rhizobiaceae; grupo de Rhizobium/Agrobacterium; Agrobacterium; Agrobacterium tumefaciens
-
imagen164 imagen165
- Agrobacterium tumefaciens str. C58 (plásmido)Agrobacterium (3 proteínas)
- ADN ligasa dependiente de ATP NP_396032
- Rhizobium leguminosarum bv. trifolii WSM1325 (plásmido)Rhizobium (10 proteínas)
- proteína del dominio de ligasa LigD de ADN polimerasa YP_002973496
- Rhizobium leguminosarum bv. trifolii WSM2304 (plásmido)Rhizobium (10 proteínas)
- proteína del dominio de ligasa LigD de ADN polimerasa YP_002278005
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| WO2012099896A3 (en) | 2012-09-20 |
| EP2665833A2 (en) | 2013-11-27 |
| DK3216878T3 (da) | 2019-06-11 |
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