RESUMEN
A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.
Asunto(s)
Codón , Escherichia coli , Regiones Promotoras Genéticas , Proteínas Recombinantes , Polimerasa Taq , Escherichia coli/genética , Escherichia coli/metabolismo , Codón/genética , Polimerasa Taq/metabolismo , Polimerasa Taq/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Thermus/genética , Thermus/enzimología , Secuencia de BasesRESUMEN
Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'â3' exonuclease domain, a 3'â5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'â3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.
Asunto(s)
Colicinas , Polimerasa Taq/genética , Polimerasa Taq/química , Polimerasa Taq/metabolismo , Colicinas/genética , Colicinas/metabolismo , Escherichia coli/metabolismo , ADN , Exonucleasas , ADN Polimerasa Dirigida por ARN/metabolismo , Thermus/genética , Thermus/metabolismoRESUMEN
Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probe the role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.
Asunto(s)
Simulación de Dinámica Molecular , Polimerasa Taq , Tirosina , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Polimerasa Taq/metabolismo , Polimerasa Taq/química , Polimerasa Taq/genética , Thermus/enzimología , Thermus/genética , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos/genética , Conformación Proteica , Especificidad por Sustrato , CinéticaRESUMEN
BACKGROUND: Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control. RESULTS: Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92. CONCLUSIONS: These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.
Asunto(s)
Nanoporos , ARN Ribosómico 16S/genética , Polimerasa Taq/genética , Genes de ARNr , Análisis de Secuencia de ADN/métodos , ADN Bacteriano/genética , Bacterias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : TâG : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : CâA : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Fosfatos , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , Reacción en Cadena de la Polimerasa , Mutación , NucleótidosRESUMEN
DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.
Asunto(s)
Inteínas , Reacción en Cadena de la Polimerasa , Ingeniería de Proteínas , Polimerasa Taq , Humanos , Inteínas/genética , Ácidos Nucleicos , Patología Molecular , Empalme de Proteína , ARN , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.
Asunto(s)
Replicación del ADN , ADN , Filogenia , Polimerasa Taq/química , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , ADN/genética , AminoácidosRESUMEN
Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.
Asunto(s)
ADN , Humanos , Polimerasa Taq/genética , Alelos , ADN/genética , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-µs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.
Asunto(s)
Replicación del ADN , Nucleótidos , Catálisis , Cinética , Nucleótidos/metabolismo , Polimerasa Taq/química , Polimerasa Taq/genética , Polimerasa Taq/metabolismoRESUMEN
Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronological overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.
Asunto(s)
Evolución Molecular Dirigida , Enzimas/metabolismo , Microfluídica/métodos , Antibacterianos/biosíntesis , Antibacterianos/química , Emulsiones/química , Enzimas/genética , Escherichia coli/química , Escherichia coli/metabolismo , Microfluídica/instrumentación , Mutagénesis , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Polimerasa Taq/genética , Polimerasa Taq/metabolismoRESUMEN
Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.
Asunto(s)
ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , Thermus/enzimología , Thermus/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Polimerasa I/química , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Polimerasa Taq/genética , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
BACKGROUND: Amyloid-ß peptide (Aß) deposition in Alzheimer's disease (AD) is due to an imbalance in its production/clearance rate. Aß is transported across the blood-brain barrier by LRP1 and P-gp as efflux transporters and RAGE as influx transporter. Vitamin D deficit and polymorphisms of the vitamin D receptor (VDR) gene are associated with high prevalence of mild cognitive impairment (MCI) and AD. Further, vitamin D promotes the expression of LRP1 and P-gp in AD-animal model brains. OBJECTIVE: To associate VDR polymorphisms Apa I (rs7975232), Taq I (rs731236), and Fok I (rs2228570) with the risk of developing MCI in a Chilean population, and to evaluate the relationship of these polymorphisms to the expression of VDR and Aß-transporters in peripheral blood mononuclear cells (PBMCs). METHODS: VDR polymorphisms Apa I, Taq I, and Fok I were determined in 128 healthy controls (HC) and 66 MCI patients. mRNA levels of VDR and Aß-transporters were evaluated in subgroups by qPCR. RESULTS: Alleles A of Apa I and C of Taq I were associated with a lower risk of MCI. HC with the Apa I AA genotype had higher mRNA levels of P-gp and LRP1, while the expression of VDR and RAGE were higher in MCI patients and HC. For Fok I, the TC genotype was associated with lower expression levels of Aß-transporters in both groups. CONCLUSION: We propose that the response to vitamin D treatment will depend on VDR polymorphisms, being more efficient in carriers of protective alleles of Apa I polymorphism.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/genética , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptores de Calcitriol/genética , Anciano , Chile/epidemiología , Disfunción Cognitiva/epidemiología , Estudios de Cohortes , Femenino , Expresión Génica , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Factores de Riesgo , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/µL of input viral genomic RNA.
Asunto(s)
COVID-19/diagnóstico , ADN Polimerasa Dirigida por ARN/química , SARS-CoV-2/aislamiento & purificación , Polimerasa Taq/química , Animales , COVID-19/genética , COVID-19/virología , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/patogenicidad , Polimerasa Taq/genéticaRESUMEN
Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein-peptide pair. Escherichia coli cells co-expressing protein-peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein-peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher-SpyTag pair and co-selecting for functionally interacting variants.
Asunto(s)
Evolución Molecular Dirigida , Escherichia coli/genética , Péptidos/genética , Mapas de Interacción de Proteínas/genética , Compartimento Celular/genética , Replicación del ADN/genética , Regulación Bacteriana de la Expresión Génica/genética , Genotipo , Luciferasas/genética , Fenotipo , Ingeniería de Proteínas , Polimerasa Taq/genéticaRESUMEN
DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
Asunto(s)
Replicación del ADN , ADN/biosíntesis , Ingeniería de Proteínas , Polimerasa Taq , ADN/genética , Polimerasa Taq/química , Polimerasa Taq/genéticaRESUMEN
In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Técnicas Biosensibles , ADN Polimerasa Dirigida por ADN/genética , Polimerasa Taq/genética , Aptámeros de Nucleótidos/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/farmacología , Humanos , Nanoestructuras/química , Conformación de Ácido Nucleico/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Polimerasa Taq/antagonistas & inhibidores , Polimerasa Taq/químicaRESUMEN
Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today's molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl ß-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/enzimología , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , Secuencia de Bases , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica/genética , Nucleótidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Polimerasa Taq/químicaRESUMEN
We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Mutación , Reacción en Cadena de la Polimerasa/métodos , Polimerasa Taq/metabolismo , Alelos , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/genética , Proteína 1 Modificadora de la Actividad de Receptores/antagonistas & inhibidores , Proteína 1 Modificadora de la Actividad de Receptores/genética , Proteína 3 Modificadora de la Actividad de Receptores/antagonistas & inhibidores , Proteína 3 Modificadora de la Actividad de Receptores/genética , Polimerasa Taq/genética , Thermus/enzimologíaRESUMEN
DNA polymerase I from Thermus aquaticus ( Taq DNA polymerase) is useful for polymerase chain reactions because of its exceptional thermostability; however, its activity at low temperatures can cause amplification of unintended products. Mutation of isoleucine 707 to leucine (I707L) slows Taq DNA polymerase at low temperatures, which decreases unwanted amplification due to mispriming. In this work, unrestrained molecular dynamics (MD) simulations were performed on I707L and wild-type (WT) Taq DNA polymerase at 341 and 298 K to determine how the mutation affects the dynamic nature of the protein. The results suggest that I707L Taq DNA polymerase remains relatively immobile at room temperature and becomes more flexible at the higher temperature, while the WT Taq DNA polymerase demonstrates less substantial differences in dynamics at high and low temperatures. These results are in agreement with previous experimental results on the I707L mutant Taq DNA polymerase that show dynamic differences at high and low temperatures. The decreased mobility of the mutant at low temperature suggests that the mutant remains longer in the blocked conformation, and this may lead to reduced activity relative to the WT at 298 K. Principal component analysis revealed that the mutation results in decoupled movements of the Q helix and fingers domain. This decoupled nature of the mutant gives way to an increasingly flexible N-terminal end of the Q helix at 341 K, a characteristic not seen for WT Taq DNA polymerase.
Asunto(s)
Frío , Simulación de Dinámica Molecular , Polimerasa Taq/química , Polimerasa Taq/metabolismo , Temperatura , Estabilidad de Enzimas , Mutación , Polimerasa Taq/genética , Thermus/enzimologíaRESUMEN
Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'â3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.